Use of recombinant B-cell differentiation factor for augmenting antibody production and stimulating bone marrow proliferation

ABSTRACT

A purified human B-cell differentiation factor (BCDF) which does not have a natural signal peptide attached to the N-terminal thereof, DNA encoding the BCDF, transformed cells containing such DNA, a process for producing and obtaining the BCDF, compositions containing the BCDF and various therapeutic uses are disclosed.

This is a continuation of application Ser. No. 07/081,746, filed on Aug.5, 1987, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to B cell differentiation factor(hereafter referred to as "BCDF") having activity in humans, a genecorresponding to human BCDF polypeptide, biotic cells havingincorporated therein the gene, a method for production of the BCDF usingthe biotic cells as well as an immunotherapeutic composition comprisinghuman BCDF as an effective component.

2. Description of the Related Art

Mature human BCDF is a material that was first discovered as a discretesubstance by the present invention. It can be widely utilized as atherapeutic composition for immunodeficiency diseases.

European Patent Application 0220574 describes certain so-calledinterferons having an amino acid sequence related to BCDF of thisinvention. However, the protein disclosed in EP 0220574 is longer thanmature human BCDF, and contains at least a signal peptide attachedthereto. The structure of mature human BCDF was not appreciated beforethe present invention. Also in contrast to the polypeptide of EP0220574, the present polypeptides do not have the anti-viral activitydescribed in EP 0220574.

Mature B cells activated by stimulation with an antigen are caused toproliferate by the aid of T cells, but it is known that one or more Tcell-derived differentiation-inducing substances are necessary tofinally differentiate B cells to reach antibody-producing cells. Thepresence of such substances has been substantiated by R. W. Dutton etal., Transplant. Rev., 23, 66 (1975) and, A. Schimpl and E. Wecker etal., Nature N. Biol., 237, 15 (1972). They have found that thesupernatant after culturing a mouse lymphocyte mixture or thesupernatant after culturing mouse lymphocytes stimulated with an antigenor mitogen can amplify a primary immune response of mouse lymphocytemass from which mouse T cells are removed or of nude mouse-derivedlymphocytes to sheep red blood cells and have given the name of T cellreplacing factor, namely TRF, to the active substance having such anactivity. Since then, TRF has been defined to be a humoral factor whichacts on B cells in such a manner that does not require any consistencyin major histocompatibility gene complex (hereafter simply referred toas MHC) non-specifically to antigen, does not induce proliferation of Bcells but induces differentiation of B cells to antibody-producingcells.

After that, functional evidence showing the presence of such a B celldifferentiation factor has been accumulated and the presence of a humandifferentiation factor analogous to the one in mice has been suggested.At present, the factor defined as described above that differentiates Bcells into antibody-producing cells has been collectively termed BCDF.

As such, BCDF plays an important role in the function of antibodyproduction of B cells in vivo in humans.

Lymphokines, which are soluble proteins having biological andpharmacological activity, act to regulate immune response mechanisms ofthe body in vivo in a trace amount. The utility of lymphokines asanti-tumor agents, an anti-viral agents, anti-bacterial agents,immunodeficiency therapeutic agents and autoimmune disease therapeuticagents has been expected due to the nature of these immunoactivesubstances (Adv. in Immunopharm., 507, 1980). BCDF in accordance withthe present invention is a lymphokine and, from this point of view, BCDFis expected to be applicable as a medical drug.

To obtain BCDF, there has been hitherto adopted a method which comprisesseparating normal T cells from human peripheral blood and stimulatingthe T cells with mitogen to produce BCDF. According to this method, manyproblems arise in that it is difficult to obtain T cells in a sufficientamount; toxic mitogens harmful to BCDF contaminate because mitogens areused and it is difficult to remove them; it is necessary to supplementserum components such as fetal bovine serum, etc. in culture of T cellsand BCDF cannot be sufficiently separated from these supplementedproteins so that failure to give pure BCDF becomes an obstacle tomedical use of BCDF. BCDF could not thus be produced industrially. Thereis also reported a method which comprises cell fusion of human T cellswith human cancer cells to give human T cell hybridomas and producingBCDF using the hybridomas (Okada et al., J. Exp. Med., 157, 583 (1983)).However, human hybridomas often tend to reduce their lymphokineproductivity during culture, so BCDF-producing human hybridomas thatwithstand practical use are still unknown.

SUMMARY OF THE INVENTION

Therefore, an object of the present invention is to provide a humanBCDF, DNA coding for the BCDF, biotic cells having such DNA and a methodfor producing such BCDF. DNA coding for human BCDF is necessary andindispensable for production of human BCDF by cells of procaryotes oreucaryotes in large quantities.

BRIEF DESCRIPTION OF DRAWINGS

In the drawings attached

FIG. 1 shows the construction of expression recombinant DNA in monkeycells.

FIG. 2 shows the BCDF activity of the culture supernatant of monkeycells (COS-7) to which PBSF 2-38 cDNA was introduced.

FIG. 3 shows the BCDF activity, as assayed by reverse plaque method, ofthe culture supernatant of monkey cells (COS-7) in which PBSF 2-38 cDNAwere introduced.

FIG. 4 illustrates the analysis of pBSF 2-38 CDNA insert b7 northernblot technique.

FIG. 5 shows the (nucleotide sequence) and amino acid sequence of BCDF.

FIG. 6 shows a restriction enzyme cleavage map. FIG. 7 illustrates theconstruction of plasmid pT13S(Nco).

FIG. 8 shows the nucleotide sequence and restriction enzyme cleavage mapof synthetic human interleukin-2 (HIL-2).

FIG. 9 shows the construction of plasmid pTBCDF-01, FIG. 10 shows theconstruction of plasmid pTBCDF-02.

FIG. 11 shows the construction of plasmid pTBCDF11.

FIG. 12 shows the construction of plasmid pTBCDF12.

FIG. 13 shows the construction of plasmid pTBCDF03.

FIG. 14 illustrates the antibody production in human B cell lineSKW6-CL4, enhanced by the introduction of human Ala-BCDF or human BCDF.

FIG. 15 illustrates that antibody production in human B cell lineSKW6-CL4 is induced by the addition of human BCDF. FIG. 15 alsoillustrates antibody production by the addition of human BCDF incombination with human IL-2.

FIG. 16 illustrates that antibody production in DBA/2 mouse spleen cellsis enhanced by the combined addition of human BCDF and human IL-2.

FIG. 17 illustrates that antibody production in the spleen cells ofBalb/c nude mouse is enhanced by the combined addition of human BCDF andhuman IL-2.

FIG. 18 illustrates that antibody production against the immunizedantigen in vivo is enhanced by the combined addition of human BCDF andhuman IL-2.

FIG. 19 illustrates that growth of DBA/2 mouse bone marrow cells isenhanced by the combined addition of human Ala-BCDF and mouse IL-3.

FIG. 20 illustrates that growth of bone marrow cells which have beeninduced by the combined addition of human Ala-BCDF and mouse IL-3 isfurther enhanced by the previous administration of lentinan.

FIG. 21 illustrates that human Ala-BCDF and mouse IL-3 stimulate theformation of colonies and clusters of DBA/2 mouse bone marrow cells.

FIG. 22 illustrates that the formation of colonies and clusters of bonemarrow cells from an immuno-deficient mouse (induced by administrationof immuno-suppressive agent) are enhanced by the combined addition ofhuman Ala-BCDF and mouse IL-3.

FIG. 23 illustrates that human Ala-BCDF induces the differentiation ofhuman myelomonocytic leukemia cell lines.

FIG. 24 indicates that recombinant BCDF induces Ig secretion ofPWM-stimulated mononuclear cells.

FIG. 25 indicates that recombinant BCDF induces Ig secretion ofPWM-stimulated B blast cells.

FIG. 26 indicates an increase of SRBC-specific antibody-producing cellnumbers of the C3H/HeJ (LPS Low Responder Mouse) spleen cells by theaddition of BCDF.

FIG. 27 indicates an increase of anti-SRBC Ig titer in serum byadministration of BCDF in vivo.

FIG. 28 indicates an augmentation of splenic hematopoiesis byadministration of BCDF in vivo.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present inventors have already discovered that human T cellstransformed by human T cell leukemia virus (hereafter referred to as"HTLV") can produce BCDF with high efficiency and obtained an authenticprotein having a B cell differentiation factor activity of 5×10⁶units/ml or more.

Production of a human BCDF-producing human T cell line can be carriedout as follows. Lymphocytes are separated from human peripheral blood,tonsil, core blood, etc. according to the density gradient centrifugalmethod, etc. using Ficoll pack, etc. and, human T cells are transformedusing HTLV in a manner similar to N. Yamamoto, Science, 217, 737 (1982).

For example, the following method can be used: 1×10⁷ /ml ofvirus-producing cell line MT-2, which is inactivated by irradiation of Xrays (12000 to 14000 rads) and 1×10⁷ / ml human lymphocytes, which areseparated by the above-mentioned method are mixed and inoculated on RPMI1640 medium containing 20% FCS, 100 μg/ml of kanamycin, 2 μg/ml ofNaHCO₃ and 25 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid(HEPES), in a plastic Petri's dish (Falcon #3003), followed by culturingat 37° C. in the presence of 5% CO₂. After culturing for 2 to 3 monthswhile replenishing a fresh medium for half of the medium twice a week,the cell line is established by the limiting dilution method. A BCDFactivity of the supernatant of the established cell is measured toobtain a cell line having the BCDF activity.

As the cells established by this method, for example, a human T cellline named VT-1(IFO 50096) can be used. There are no particularconditions for growing VT-1 but culture conditions conventionally usedmay be appropriately adopted. BCDF can also be produced in aconventional manner but it is preferred to perform the production in aprotein-free medium.

As a medium most suited for enhancing the growth of VT-1, there is useda medium supplemented with, for example, 1 to 30%, preferably 20% ofFCS. A medium most suited for the production of BCDF may be theaforesaid FCS-free medium. The survival rate of VT-1 is maintained 70%or more even after culturing 48 hours in a FCS-free complete syntheticmedium.

In the case where BCDF is produced from T cells, it was essentiallyrequired in the prior art to supplement protein such as FCS or mitogento media (cf., T. Teranishi et al., J. Immunol., 128, 1093 (1982), A.Muraguchi et al., J. Immunol., 127, 412 (1981)). To the contrary, in thecase where human BCDF is produced using VT-1, it is notable that it isneither necessary to supplement serum such as FCS, protein components inblood and other proteinaceous components to media nor necessary tosupplement even mitogen to T cells or B cells. Therefore, not only canBCDF be produced at low costs without using expensive FCS but also safeBCDF free from heterogeneous proteins or mitogen harmful to the humanbody can easily be obtained.

The aforesaid method for producing BCDF using VT-1 can be performedunder various environmental conditions. However, the VT-1 culture shouldbe preferably maintained in moisture controlled air containingapproximately 5 to 10% of carbon dioxide in a temperature range ofapproximately 35° to 38° C. Further, the pH of the medium should ideallybe maintained under slightly alkaline conditions of approximately 7.0 to7.4. VT-1 is inoculated on various types of incubators such as a roundbottom microplate, etc. in various volumes such as 100 μl, etc. Tissueculture flasks such as Flask No. 3013 or No. 3024 marketed from FalconLabware, Div. Becton, Dickinson and Co. can also be used. As anothermethod, roller bottles such as Bottle No. 3027 marketed from theaforesaid Falcon Labware can also be used as incubators.

An initial density of the cells for the optimum conditions for culturingVT-1 and growing the cell count is 1×10⁴ to 5×10⁵ preferably 2×10⁵. WhenVT-1 is cultured under the conditions described above, the cell densityincreases from approximately 5×10⁵ to 2×10⁶ per 1 ml of the medium,generally after 2 to 7 days and therefore, a fresh medium is againreplenished to reduce the cell density to 1×10⁴ to 5×10⁵ per 1 ml of themedium and the incubation is again continued. After continuing theincubation of VT-1 to reach the desired cell number as above, the cellsare separated by centrifugation, etc. After the cells are rinsed with aprotein-free complete synthetic medium, they are inoculated on a freshcomplete synthetic medium. In this case, it is preferred that theinitial density of the cells be approximately 1×10⁴ to 1×10⁷ per 1 ml ofthe medium, ideally 1×10⁶ cells per 1 ml of the medium.

The quantity of BCDF produced by culturing VT-1 varies with passage oftime. When VT-1 is cultured in RPMI 1640 medium (containing 100 units/mlof penicillin, 100 μg/ml of streptomycin, 10 μg/ml of gentamycin and 16mM of NaHCO₃), for example, in an initial cell density of 1×10⁶ /ml, theBCDF activity reaches its peak level after 48 hours. Further, the BCDFactivity present for a subsequent 24 hours decreases slightly. As such,the optimum incubation time for producing BCDF by VT-1 in RPMI 1640medium is approximately 24 to 78 hours.

BCDF can be concentrated and purified from the aforesaid culturesupernatant by various methods such as salting out, lyophilization,ultrafiltration, gel filtration chromatography, ion exchangechromatography, affinity chromatography, chromatofocusing, reversedphase chromatography, focusing electrophoresis, gel electrophoresis,etc.

Turning next to measurement of the BCDF activity, the following methodsmay be mentioned.

BCDF activity is measured using human B cell line CL4 producing IgM inresponse to human BCDF (T. Hirano et al., Proc. Natl. Acad. Sci., 82,5490 (1985)). A sample solution for assaying a BCDF activity and 6×10³cells of CL4 are added to 200 μl of 10% FCS-containing RPMI 1640 medium(containing 100 units/ml of penicillin, 100 μg/ml of streptomycin, 10μg/ml of gentamycin and 16 mM of NaHCO₃). The mixture is cultured at 37°C. for 3 days in the presence of 5% CO₂ in a 96 well microplate, wherebythe amount of IgM in the supernatant is measured by enzyme immunoassay.Under these conditions, the activity of BCDF showing 50% of the maximumIgM production amount (the highest CL4 reaction) is defined to be 1U/ml.

The BCDF activity can also be measured by the reverse PFC method usinghuman B cell line CL4 producing IgM in response to human BCDF (O. Saikiet al., Eur. J. Immunol., 13, 31 (1983)). A sample solution for assayinga BCDF activity and 1×10⁴ cells of CL4 are added to 200 μl of 10%FCS-containing RPMI 1640 medium (additives are the same as describedabove). The mixture is cultured at 37° C. for 3 days in the presence of5% CO₂ in a 96 well microplate. The culture cells, complement,anti-human IgM antibody and protein A-bound sheep red blood cells aremixed with agarose dissolved in Hanks' solution and the mixture isspread and solidified on a Petri's dish. After culturing overnight at37° C. in the presence of 5% CO₂, the number of cells differentiatedinto IgM-producing cells by the action of BCDF is measured by the numberof hemolytic plaques formed.

To identify and collect the gene coding for human BCDF, RNA is extractedand harvested from the VT-1 cells cultured under the optimum conditionsfor the production of BCDF described above to make a cDNA library andcDNA coding for BCDF is cloned from the library. Therefore, the presentinventors have identified BCDF cDNA as will be later shown in theexamples by completely purifying human BCDF produced by the VT-1 cells,determining the amino acid sequence of BCDF at the N-terminal thereof,and the amino acid partial sequence of the fragment peptides which areobtained by restricted fragmentation of lysyl endopeptidase,synthesizing oligonucleotides corresponding to the respective peptides,and screening clones complementarily hybridized with some syntheticprobes from the aforesaid cDNA library using the thus synthesizedoligonucleotide probes. The base sequence of the thus obtained cDNA hasbeen determined in a conventional manner and the gene coding for BCDFhas been established. From the sequence of this gene, it has been foundthat human BCDF is a polypeptide composed of 184 amino acids. At thesame time, in order to express the thus obtained cDNA in eucaryoticcells, the cDNA has been connected with an expression vector and itsgene has been introduced into the cells. BCDF is allowed to be producedin the supernatant by incubation of the cells, and pure human BCDF isobtained by purification operations. Recombinant human BCDFcorresponding to the genetic structure of BCDF has also been produced,and it has been found that this BCDF has the same physicochemical andbiological properties as those of the VT-1 cells or human BCDF obtainedfrom human cells. From the foregoing, it has been finally proven thatthe identified gene does code for human BCDF protein.

On the other hand, human BCDF can also be produced in procaryotes.Namely, the gene coding for human BCDF is introduced into vector DNA toexpress the same, the thus obtained recombinant DNA is introduced in aprocaryotic host and the obtained transformed microorganism is cultured.

The gene coding for BCDF has at least amino acid sequence (I) or (II)(see below) or the base sequence corresponding to its partially modifiedstructure. Host cells of the procaryote in which the recombinant DNA isintroduced may be Escherichia coli, Bacillus subtilis and othermicroorganisms, which are obvious to one skilled in the field of currentgenetic engineering.

Media and methods for culturing the transformed microorganisms(procaryotes) may be conventional media and methods.

In the case where human BCDF is accumulated in the transformedmicroorganism, BCDF can be recovered and purified by a method which oneskilled in the art can readily perform. Simply stated, the cells arecollected by centrifugation after incubation and suspended in a solutioncontaining lysozyme or a solution containing a detergent; aftercompletion of the reaction, freezing and thawing are repeated to obtainthe cell extract and the extract can be purified by the aforesaidpurification method and/or in a simple manner such as affinitychromatography using an anti-BCDF antibody-immobilized column.

At this time, however, there is no report on a method for producing BCDFby procaryotes such as Escherichia coli, etc. namely, a method forproducing BCDF by procaryotes which comprises incorporating DNAcorresponding to BCDF into a procaryotic vector, replicating,transcribing and translating in the procaryotic cells, and BCDF producedby such a method.

As a result of further extensive investigations to solve the foregoingproblems, the present inventors have accomplished the present inventionby culturing procaryotic cells transformed by recombinant DNA composedof vector DNA capable of replicating the gene coding for the polypeptidehaving human BCDF activity and procaryotic cells and harvesting theproduced human BCDF. Hereafter this invention will be described indetail.

The present inventors have already identified a gene coding for humanBCDF, paying attention to the fact that VT-1 cells (IFO 50096) or ahuman T cell transformed by HTLV (human T cells leukemia virus) producehuman BCDF in large quantities (Japanese Patent Application No.184858/86).

Details of the cloning procedure of the gene coding for human BCDF aredescribed in the example given later.

Turning now to production of the procaryote producing human BCDF, thegene coding for human BCDF is incorporated into vector DNA capable ofreplicating in procaryotic cells. In this case, DNA coding for humanBCDF may be inserted downstream from the promoter sequence of theexpression vector; alternatively, a DNA fragment having the promotersequence may be inserted before or after the insertion of cDNA of theexpression vector and upstream from the DNA coding for human BCDF. Inthis case, cDNA coding for human BCDF codes for a polypeptide composedof 212 amino acids as shown in FIG. 5, but the hydrophobic N-terminalregion corresponding to 28 amino acids of this polypeptide is a signalsequence, which is cleaved during the course of secretion. Accordingly,it is desired to express the cDNA portion coding for mature human BCDFpolypeptide composed of 184 amino acids starting from the N-terminalPro.

To construct the expression plasmid, the vector is cleaved with anappropriate restriction enzyme and if necessary and desired, insertedwith an appropriate linker or an oligonucleotide adapter capable ofannealing when combined. The thus finished double stranded DNA is mixedwith vector DNA, and the mixture is ligated using ligase.

The thus obtained recombinant DNA is introduced in a procaryotic hostand a human BCDF-producing strain can be selected from the obtainedtransformed micro-organisms.

As the procaryote in the present invention, Escherichia coli, Bacillussubtilis, etc. can be employed; it is preferred to use Escherichia coli.

Examples of the vectors for Escherichia coli which can be used in thepresent invention include EK type plasmid vectors (stringent type;pSC101, pRK353, pRK646, pRK248, pDF41, etc.), EK type plasmid vectors(relaxed type: ColE1, pVH51, pAC105, RSF2124, pCR1, pMB9, pBR313,pBR322, pBR324, pBR325, pBR327, pBR328, pKY2289, pKY2700, pKN80, pKC7,pKB158, pMK 2004, pACYC1, pACYC184, dul, etc.), λgt type phase vectors:λgt.λc. λgt.λB, λWES, λC, λWES, λB, λZJvir., λB', λALO, λB, λWES.Ts622,λDam, etc.

Further as the promoters, all promoters that function in Escherichiacoli, including trp, lac, tac, tufB, B-lactamase and lpp promoter, maybe used.

For the transformation of host cells using recombinant DNA, thefollowing method is conventionally used. In the case where a procaryotesuch as Escherichia coli is a host, competent cells capable ofincorporating this DNA can be transformed by the well known CaCl₂method, after recovering cells in exponential growth phase. Presence ofMgCl₂ or RbCl in the transformation reaction solution improves thetransformation efficiency. It is also possible to perform thetransformation after preparation of protoplast of the host cell.

The thus obtained recombinant procaryote cell in which the human BCDFgene has been incorporated may be cultured in a conventional manner.

The thus obtained BCDF can be concentrated and purified from theaforesaid culture supernatant by various methods such as salting out,lyophilization, ultrafiltration, gel filtration chromatography, ionexchange chromatography, affinity chromatography, chromatofocusing,reverse phase chromatography, focusing electrophoresis, gelelectrophoresis, etc.

In case the human BCDF is accumulated in the transformed microorganism,BCDF can be recovered and purified by conventional methods. Simplystated, the cells are collected by centrifugation after incubation andsuspended in a solution containing lysozyme or a solution containing adetergent; after completion of the reaction, freezing and thawing arerepeated to collect the cell extract and the extract can be purified bythe aforesaid purification method and/or in a simple manner such asaffinity chromatography using anti-BCDF antibody-immobilized column.

Turning next to measurement of the BCDF activity, the following methodsmay be mentioned.

The BCDF activity is measured using human B cell line CL4 producing IgMin response to human BCDF (T. Hirano et al., Proc. Natl. Acad. Sci., 82,5490 (1985)). A sample solution for assaying a BCDF activity and 6×10³cells of CL4 are added to 200 μl of 10% FCS-containing RPMI 1640 medium(containing 100 units/ml of penicillin, 100 μg/ml of streptomycin, 10μg/ml of gentamycin and 16 mM of NaHCO₃). The mixture is cultured at 37°C. for 3 days in the presence of 5% CO₂ in a 96 well microplate, wherebythe amount of IgM in the supernatant is measured by enzyme immunoassay.Under these conditions, the activity of BCDF showing 50% of the maximumIgM production amount (the highest CL4 reaction) is defined to be 1U/ml.

The BCDF activity can also be measured by the reverse PFC method usinghuman B cell line CL4 producing IgM in response to human BCDF (O. Saikiet al., Eur. J. Immunol., 13, 31 (1983)). A sample solution for assayinga BCDF activity and 1×10⁴ cells of CL4 are added to 200 μl of 10%FCS-containing 1640 medium (additives are the same as described above).The mixture is cultured at 37° C. for 3 days in the presence of 5% CO₂in a 96 well microplate. The culture cells, complement, anti-human IgMantibody and protein A-bound sheep red blood cells are mixed withagarose dissolved in a Hanks' solution and the mixture is spread andsolidified on a Petri's dish. After culturing overnight at 37° C. in thepresence of 5% CO₂, the number of cells differentiated intoIgM-producing cells by the action of BCDF is measured by the number ofhemolytic plaques formed.

As is also known with human interferon genes, eucaryotic genes showpolymorphism (Taniguchi et al., Gene, 10, 11-15 (1980), Ohno andTaniguchi, Proc. Natl. Acad. Sci., USA, 77, 5305-5309 (1981), Gray etal., Nature, 295, 501-508 (1981)). As a result of polymorphism, someamino acids of the protein product may be replaced in some cases andother cases, nothing changes even though base sequences may change.Accordingly, the present invention also includes polypeptide obtained byreplacing one or more amino acids for one or more amino acids in theamino acid sequence of FIG. 5 and DNA coding for this polypeptide, aslong as they possess the human BCDF activity. For example, a cysteineresidue may be replaced by a neutral amino acid, if the cysteine residuewhich is replaced is not required for BCDF activity. Further as will beshown in the examples, the present invention also includes polypeptidehaving human BCDF activity deficient in one or more amino acids,polypeptides having added thereto one or more amino acids, polypeptideshaving combined sequences thereof (including replacement with aminoacids) and DNAs having base sequences coding for these polypeptides.Even though there is a modified region having a supplemental amino acidsequence that inhibits the polypeptide function as human BCDF, such canbe utilized as the polypeptide and gene of the present invention insofaras the newly added region can be readily removed. Namely, anypolypeptide having human BCDF activity is as described in thisapplication human BCDF in accordance with the present invention.

Application of the thus produced BCDF to the clinical field is roughlyclassified into three areas. First, anti-BCDF antibody is produced byBCDF; the antibody can be used for analysis of immunological state usingthe immunoassay system of BCDF with BCDF and anti-BCDF antibody and atthe same time, can be used for repair of functional abnormality of Bcells sometimes noted in autoimmune diseases. Second, anotherapplication is to therapy for various diseases. For example, theantibody-producing function can be made normal by administrating BCDFalone or together with other lymphokines or immunotherapeutic agents, inpatients with immunodeficiency due to reduced antibody producingactivity of B cells accompanied by reduced helper function of T cells.

Further noting the differentiation activity of BCDF, BCDF can also beused as an anti-malignant tumor agent by differentiating malignant tumorcell lines by BCDF and causing growth inhibition.

As still a further application of BCDF, the following is considered. Itis reported that normal B cells can be cultured over a long period oftime by supplementing B cell growth factor (BCGF) (K. Yoshizaki et al.,J. Immunol., 130, 1241 (1983)) and other T cell factors includinglymphokines to media (cf. B. Sredni et al., J. Exp. Med., 154, 1500(1981)). By allowing BCDF to act on these normal B cells cultured or Bcells transformed by EB virus in an appropriate period of time, antibodycan be produced in vitro. By cloning B cells producing antibody whichrecognizes a specific antigen present on the surface of, for example,pathogenic bacteria, pathogenic virus, pathogenic protozoa, cancercells, etc. to monoclone and culturing the cloned normal B cells or thecells transformed by EB virus in combination with BCDF and otherlymphokines, useful monoclonal antibodies can be produced. Theseantibodies can be utilized for therapy and diagnosis of infectiousdiseases and cancers.

As described above, BCDF is a substance effective over an extremely widerange.

The present invention also relates to an immunotherapeutic compositioneffective for therapy of primary and secondary immunodeficiencies,infectious diseases with bacteria, fungi, viruses, protozoa, etc.,cancers and for augmentation of haemopoietic function. Moreparticularly, the present invention relates to an immunotherapeuticcomposition comprising human BCDF as an effective component.

As such, BCDF plays an important role in the function of antibodyproduction of B cells in vivo in humans. With respect to human BCDFplaying such an important role, the present inventors have madeextensive investigations and succeeded in determination of its DNAsequence and amino acid sequence (Japanese Patent Application Nos.184858/86 and 200433/86) and in production of human BCDF usingEscherichia coli (Japanese Patent Application No. 302699/86).

However, no report has been hitherto made on pharmacological effects ofthis human BCDF against cancer, infectious diseases, primaryimmunodeficiency or secondary immunodeficiency, for example, reductionin the number of leucocytes observed upon chemotherapy, and radiationtreatment of patients with malignant tumors.

Now, cancers, immunodeficiency and infectious diseases in associationtherewith for which the immunotherapeutic composition comprising humanBCDF is effective, will be briefly described and at the same time,current therapy will be described briefly.

In recent years, it has been proposed to call human BCDF BSF-2 (Nature,324, 73 (1986)) but the term BCDF is used in this specification.

Immunodeficiency syndromes collectively refer to a state in which anyaspect of the immune system is deficient and host defense ability of thebody is reduced.

Immunodeficiency syndromes are roughly classified into two classes:primary immunodeficiency which is considered to be based on a congenitalcause, and secondary immunodeficiency which is considered to be causedby any external cause or in association with other diseases.

In most cases, causes for primary immunodeficiency diseases are geneticdefects of T cells and/or B cells. On the other hand, causes forsecondary immunodeficiency diseases are various, but one of the majorcauses is infection with bacteria, viruses, etc.

Further, secondary immunodeficiency diseases are sometimes causedbecause the number of leucocytes is markedly reduced due to use ofanti-cancer agents, radio-therapy, etc. When one suffers fromimmunodeficiency diseases, host defense ability of the living bodydecreases so that infectious diseases are frequently induced. Mostpathogens of the infectious diseases are those that have been harmlessheretofore but exhibit pathogenicity based on a change in the livingbody; such infectious diseases are called opportunistic infections. Suchinfectious diseases are noted with high frequency upon administration ofanti-cancer agents, in particular, upon chemotherapy of acute leukemiaand transplantation of bone marrow and, a lethal rate is also high. Oneopportunistic infection is carinii pneumonia induced by cariniiprotozoa, which is found in the terminal stage of AIDS.

These primary and secondary immunodeficient diseases are treated by thefollowing 3 methods.

(1) Administration of chemotherapeutic agents such as antibiotics,antiviral agents (for example, AZT in the case of AIDS)

(2) Administration of human immunoglobulin or vaccine

(3) Use of both in combination

However, when the immune function is decreased, the above treatments donot exhibit pronounced effects. In addition, the therapeutic agentsdescribed above involve serious drawbacks shown below.

Firstly, antibiotics and antiviral agents are accompanied by seriousside effects. In the case of antibiotics, resistant bacteria appear ormicrobial substitution occurs so that the effectiveness is restricted.Further in the case of immunoglobulin preparations, the amount ofantibody to the objective infectious bacteria is only a trace so thatthe effect is poor. Further, with vaccines, the effect is hardly notedin an immunodeficient state.

Further as an example of secondary immunodeficiency diseases, whenchemotherapeutic agents are administered to the patient with cancer andhematopoietic function is thus reduced to lead to a readily infectiousstate, the aforesaid therapeutic agents do not have any action ofrecovering the reduced hematopoietic function or differentiating andinducing the cancer cells themselves into normal cells. Accordingly,fundamental treatment is impossible.

Therefore an object of the present invention is to provide therapeuticcompositions for treatment of cancers, primary and secondaryimmunodeficiency diseases as well as various infectious diseases inducedby immunodeficiency diseases which are presently unknown and haveactions in combination that (1) side effects are minimized, (2) there isno problem of appearance of resistant bacteria or microbialsubstitution, (3) antibody production specific to the objective antigenis induced and strengthened, (4) hematopoietic function is recovered,and (5) differentiation and induction of cancer cells is obtained, andthe like.

As a result of extensive investigations to solve the foregoing problems,the present inventors have found that immunotherapeutic compositionscomprising human BCDF as an effective component are effective forcancers, primary and secondary immunodeficiency diseases and variousinfectious diseases induced thereby and have accomplished the presentinvention.

Namely, the present invention relates to an immunotherapeuticcomposition comprising human BCDF as an effective component. The humanBCDF in accordance with the present invention has, for example, aminoacid sequence (I) or (II) described below:

    __________________________________________________________________________    Amino acid sequence (I):                                                      __________________________________________________________________________    PRO VAL PRO PRO GLY GLU ASP SER LYS ASP VAL                                   ALA ALA PRO HIS ARG GLN PRO LEU THR SER SER                                   GLU ARG ILE ASP LYS GLN ILE ARG TYR ILE LEU                                   ASP GLY ILE SER ALA LEU ARG LYS GLU THR CYS                                   ASN LYS SER ASN MET CYS GLU SER SER LYS GLU                                   ALA LEU ALA GLU ASN ASN LEU ASN LEU PRO LYS                                   MET ALA GLU LYS ASP GLY CYS PHE GLN SER GLY                                   PHE ASN GLU GLU THR CYS LEU VAL LYS ILE ILE                                   THR GLY LEU LEU GLU PHE GLU VAL TYR LEU GLU                                   TYR LEU GLN ASN ARG PHE GLU SER SER GLU GLU                                   GLN ALA ARG ALA VAL GLN MET SER THR LYS VAL                                   LEU ILE GLN PHE LEU GLN LYS LYS ALA LYS ASN                                   LEU ASP ALA ILE THR THR PRO ASP PRO THR THR                                   ASN ALA SER LEU LEU THR LYS LEU GLN ALA GLN                                   ASN GLN TRP LEU GLN ASP MET THR THR HIS LEU                                   ILE LEU ARG SER PHE LYS GLU PHE LEU GLN SER                                   SER LEU ARG ALA LEU ARG GLN MET                                               __________________________________________________________________________

    __________________________________________________________________________    Amino acid sequence (II):                                                     __________________________________________________________________________    ALA PRO VAL PRO PRO GLY GLU ASP SER LYS ASP                                   VAL ALA ALA PRO HIS ARG GLN PRO LEU THR SER                                   SER GLU ARG ILE ASP LYS GLN ILE ARG TYR ILE                                   LEU ASP GLY ILE SER ALA LEU ARG LYS GLU THR                                   CYS ASN LYS SER ASN MET CYS GLU SER SER LYS                                   GLU ALA LEU ALA GLU ASN ASN LEU ASN LEU PRO                                   LYS MET ALA GLU LYS ASP GLY CYS PHE GLN SER                                   GLY PHE ASN GLU GLU THR CYS LEU VAL LYS ILE                                   ILE THR GLY LEU LEU GLU PHE GLU VAL TYR LEU                                   GLU TYR LEU GLN ASN ARG PHE GLU SER SER GLU                                   GLU GLN ALA ARG ALA VAL GLN MET SER THR LYS                                   VAL LEU ILE GLN PHE LEU GLN LYS LYS ALA LYS                                   ASN LEU ASP ALA ILE THR THR PRO ASP PRO THR                                   THR ASN ALA SER LEU LEU THR LYS LEU GLN ALA                                   GLN ASN GLN TRP LEU GLN ASP MET THR THR HIS                                   LEU ILE LEU ARG SER PHE LYS GLU PHE LEU GLN                                   SER SER LEU ARG ALA LEU ARG GLN MET                                           __________________________________________________________________________

Amino acid sequence (I) is natural human BCDF and amino acid sequence(II) is a polypeptide having one Ala added to the N-terminal of naturalhuman BCDF (hereafter referred to as Ala-BCDF). However, it is notalways necessary that human BCDF used in the present invention takes thestructure shown by amino acid sequence (I) or amino acid sequence (II)described above.

Namely, that having a structure in which one or more amino acids areadded from the N terminal and/or C terminal of natural human BCDF andthat having a structure in which one or more amino acids in thestructure of natural human BCDF are replaced with other amino acids canalso be used as human BCDF of the present invention as far as they havehuman BCDF activity. Natural human BCDF or human Ala-BCDF is preferablyemployed. The content of human BCDF in accordance with the presentinvention is 0.0001 to 100 wt %, preferably 0.1 to 1.0 wt %, based onthe immunotherapeutic composition.

The immunotherapeutic composition comprising human BCDF as an effectivecomponent of the present invention may also contain a stabilizer such asserum albumin, etc., an excipient such as mannitol, etc. In addition tohuman BCDF, the immunotherapeutic composition of the present inventionmay further contain at least one of human IL-2, human IL-3, lentinan andmouse IL-3 as an aid.

When such an aid is incorporated, the effect of the immunotherapeuticcomposition increases synergistically. While there is no particularlimitation to the amount of these aids to be incorporated, it ispreferred to incorporate 0.0001 to 200,000 wt %, based on human BCDFbeing 100. The amount of these aids to be added is not limited to therange described above but may be approximately determined depending uponcondition, age of the patient, etc.

Aids such as human IL-2, human IL-3, mouse IL-3 and lentinan are notalways given simultaneously with human BCDF as the same composition.Namely, these aids may also be administered in an appropriate periodprior to or after administration of the immunotherapeutic compositioncomprising human BCDF as the effective component.

Of course, the immunotherapeutic compositions of the present inventionmay also be used in combination with other chemotherapeutic agents,anti-cancer agents, anti-viral agents, antibiotics, etc. Theimmunotherapeutic composition may also be used in combination withvaccine therapy to strengthen the effect. The immunotherapeuticcomposition may be given by intravenous injection, intramuscularinjection or subcutaneous injection. That is, the composition may begiven in any form of injection.

Human BCDF used in the present invention may be produced from human Tcells, B cells, fibroblasts, etc. and purified in a known manner (Proc.Natl. Acad. Sci., USA, 82, 5490 (1985)) or produced by culturing astrain obtained by transformation of a gene encoding human BCDF to anappropriate host such as Escherichia coli, yeast, monkey cells (COScells), hamster cells, etc. using an appropriate vector and furtherpurified. With respect to the production of human BCDF, its process willbe explained in the examples again.

It is clear that the immunotherapeutic composition of the presentinvention is effective for treatment and prophylaxis of variousinfectious diseases (carinii pneumonia, etc. induced from AIDS) derivedby immunodeficient diseases. For example, in case that pathogens such asbacteria, fungi, protozoa, viruses, etc. are infected, an antibodythereto is produced and then the antibody protects the living body fromthe pathogens by 1) direct neutralization and precipitation, 2)augmentation of phagocytosis of phagocytes by opsonification, 3) lysisby activation of the complement system, 4) antibody-dependent cellmediated cytotoxicity, etc.

Namely, the immunotherapeutic composition of the present invention canenhance production of specific antibody by the patient with primaryimmunodeficiency diseases due to genetic factors, etc. and secondaryimmunodeficiency diseases in which immune function is reduced due toadministration of chemotherapeutic agents or administration ofimmunosuppressive agents and viral infections, etc., whereby a readilyinfectious state can be improved and treated.

The present inventors have found that human BCDFs not only enhanceantibody production but also effect growth of bone marrow cells, as wellas induction of differentiation of tumor cells. From these actions, ithas been noted that BCDF is not only effective for treatment ofinfectious diseases due to enhanced antibody production to the patientwith primary immunodeficiency diseases or with secondaryimmunodeficiency diseases but can also accelerate hematopoietic functionin the patient with immunodeficiency diseases and exhibit therapeuticeffects in the patient with cancer.

It is already known that 1) IL-2 is capable of enhancing antibodyproduction, 2) colony stimulating factor (CSF) causes growth of bonemarrow cells and 3) γ-interferon (γ-IFN) has an action of induction ofdifferentiation of tumor cells, but there is currently unknown asubstance having three functions of (1) enhancement of antibodyproduction, (2) growth of bone marrow cells and (3) induction ofdifferentiation of tumor cells in combination. Accordingly, human BCDFin accordance with the present invention is an important drug which wasunknown until now.

The immunotherapeutic composition comprising human BCDF as the effectivecomponent of the present invention has three functions of (1)enhancement of antibody production (2) growth of bone marrow cells and(3) induction of differentiation of tumor cells in combination so thatthe immunotherapeutic composition is effective for treatment of primaryimmunodeficiency diseases, secondary immunodeficiency diseasesaccompanied by viral infections or administration of anti-cancer agentsor immunosuppressive agents, as well as the cancer patient.

Further in the patient with immunodeficiency diseases, antibodyproduction is decreased as a matter of course and various infectiousdiseases have been caused or are ready to be caused due to decreasedhematopoietic function. The immuno-therapeutic composition comprisinghuman BCDF as the effective component of the present invention is alsoeffective for treatment and prophylaxis of these infectious diseases.

The present inventors described the synergistic effect of IL-2 or IL-3with BCDF in antibody production and hematopoiesis, especially. Butcytokines, having synergistic effect with BCDF, are not limited only toIL-2 and IL-3. Accordingly, it is supposed that BCDF will actsynergistically with other cytokines, such as IL-1, IL-4, IL-5, BCGF,GM-CSF, G-CSF, M-CSF, TNF, MAF, etc., in many biological functionassays. For example, results have been obtained by the present inventorswhich show that IL-1 exhibits a synergistic effect in differentiation oftumor cells with BCDF. Other cases are shown below.

1) IL-4, IL-5 or BCGF for synergistic augmentation of antibodyproduction.

2) IL-1 or TNF for synergistic augmentation of differentiation of tumorcells.

3) CSFs, as well as IL-3, for synergistic augmentation of cellproliferation.

4) MAF, as well as LNT (lentinan), for partial augmentation of hostdefense mechanisms.

Thus, immunotherapeutic compositions containing BCDF and one or morecytokines are also useful for treatment of infection, etc., byaugmentation of host defense mechanisms.

The invention now being generally described, the same will be betterunderstood by reference to certain specific examples which are includedherein for purposes of illustration only and are not intended to belimiting of the invention or any embodiment thereof, unless specified.

EXAMPLE 1

RPMI 1640 medium (1 l) (glutamine (2 mM), 2 ME (5×10⁻⁵ M), penicillin(100 units/ml), streptomycin (100 μg/ml), gentamycin (20 μg/ml), NaHCO₃(16 mM)) which further contains 20% FCS was placed in a 2 l plasticroller type fermentor (roller culture bottles) (Falcon #3027)(hereinafter referred to as "roller"). Cells (2×10⁵ /ml) of VT-1 wereinoculated in the medium in the roller and cultured at 37° C. at 8 rpmfor 3 days. After the cultivation, the cultured mixture was centrifugedto collect the cells which were then washed twice with RPMI 1640 medium.The VT-1 cells thus prepared were suspended at a cell concentration of1×10⁶ /ml in RPMI 1640 medium (1 l) in a 2 liter-roller. The suspensionthus obtained was cultured at 37° C. at 8rpm for 2 days. After thecultivation, the mixture was centrifuged to obtain a supernatantsolution.

The supernatant containing BCDF, prepared as above from the culture ofVT-1, was treated in a manner described below, so that BCDF was isolatedin a purified form. Thus, the supernatant, freed from the cells (10 l),was filtered under a pressure of 4 Kg/cm² in a nitrogen atmospherethrough an ultrafiltration device (Amicon cell Type No. 2000 for a largescale treatment (a mass treatment): a product of Amicon Corporation;Massachusetts, USA) furnished with an ultrafiltration membrane (AmiconYM-10, Amicon Corporation, a product of Amicon Corporation;Massachusetts, USA). A residual concentrate (100 ml) remaining over theultrafiltration membrane was again filtered under a pressure of 4 kg/cm²in a nitrogen atmosphere through an ultrafiltration equipment (Amiconstandard cell Type No. 52) furnished with an ultrafiltration membraneAmicon YM-10. The residual concentrate (5 ml) over the ultrafiltrationmembrane was collected.

The concentrated supernatant was subjected to gel-filtration on a column(LKB producer, Sweden, 2.6×90 cm) filled with AcA-34 gel which hadpreviously been equilibrated in PBS ((0.01M) phosphate buffer (pH 7.0)containing sodium chloride (0.15M)). The AcA-34 gel in the column waseluted with PBS, the eluate being fractionated into 5 ml fractions. BCDFactivity of these fractions was examined. The fractions having BCDFactivity were found to be in the fractions corresponding to molecularweight of 3.5±0.5×10⁴ Dalton. The gel-filtration column was assayed bythe use of a molecular weight marker (product of Pharmacia FineChemicals; Sweden) in the following manner.

The fractions containing blue-dextran 200 (2×10⁶), ferritin (4.5×10⁵),aldolase (1.58×10⁵), ovalbumin (4.5×10⁴), chymotrypsinogen (2.5×10⁴) andcytochrome C (1.17×10⁴) or BCDF were collected and combined, and thebuffer solution of the combined fractions was converted into a 25mMpiperazine-HCl buffer solution (pH 6.3) on ultrafiltration equipmentfurnished with an ultrafiltration membrane (Amicon YM-10). The BCDFfractions, as obtained in AcA-34 gel column chromatography, were passedthrough a column, filled with Mono P (Pharmacia Fine Chemicals, Sweden)which had previously been equilibrated in 25 mM piperazine-HCl buffersolution (pH 6.3). The column filled with the Mono P, was washed with 25mM piperazine-HCl buffer solution, followed by elution with 40 ml) ofpoly-buffer 74 (Pharmacia Fine Chemicals, Sweden) which had been diluted10 times and adjusted to pH 4.5 with aqueous HCl. The elution wasperformed at a flow rate of the eluent as 0.5 ml/minute by means ofFirst protein Liquid Chromatography (FPLC) (Pharmacia Fine Chemicals,Sweden). The eluate was fractionated into 1 ml portions, on which BCDFactivity and pH value were examined. The BCDF activity was detected inthe fractions of eluate at the pH value 4.9 to 5.1.

The BCDF active fraction obtained in the chromatography on the column ofMono P was subjected to high performance liquid chromatography on areverse phase chromatography column; Synchropak RPP (C18) (250×4.1 mm,Synchrom) which had been buffered with 0.1% aqueous TFA (trifluoroaceticacid). The column was eluted by gradient elution while increasingacetonitrile concentration from 0 to 60% in 0.1% aqueous TFA solution(eluting solution). The fraction of eluate which was eluted out at theconcentration of acetonitrile 50 to 55% indicated a peak of absorption(O.D. 280) as separated distinctly from the other peaks of absorption(O.D. 280). The BCDF activity was detected correspondingly to andcoincidently with this peak. This fraction was then lyophilized toobtain a BCDF preparate.

The BCDF preparate was subjected to electrophoresis on SDS-polyacrylicamide gel (12%) under a reducing condition. After the electrophoresis,the gel fractions corresponding to molecular weight of 21000 wasseparated from other gel fractions by cutting. The fractions isolatedwas admixed with SDS (0.05%) and NH₄ HCO₃ in an Eppendorf tube and themixture was stirred at 37° C. over-night so that BCDF was extracted. Theextract thus obtained was again subjected to HPLC on reverse phasechromatography column; Synchropak RP-P (C18) (250×4.1 mm, Synchrom)which was eluted by gradient elution while increasing acetonitrileconcentration from 0 to 60% in 0.1% aqueous TFA solution. The fractionof eluate which was eluted at a concentration of acetonitrile of 50 to55% indicated a peak of absorption (O.D. 280) as separated distinctlyfrom the other peaks (O.D. 280). It was found that the peak correspondedto the BCDF activity. This fraction was lyophilized to obtain a purifiedBCDF preparate.

In determination of amino acid sequence in BCDF protein, the purifiedBCDF (6 μg) obtained as above was introduced into a protein sequencer(See: Applied Biosystem Co., Calif, Model 470 A). The determination ofamino acid sequence was performed according to the method described inJ. Biol. Chem., 193, 265-275 (1951). The sequence of amino acidsstarting from the N-terminal was as follows;

    ______________________________________                                        Pro    Val        Pro    Pro     Gly  Glu                                     Asp    Ser        Lys    Asp     Val  Ala                                     Ala                                                                           ______________________________________                                    

EXAMPLE 2

A purified BCDF preparation (20 μg), prepared the same manner as inExample 1, was dissolved in 5 mM Tris-HCl buffer solution (pH 9.5), towhich was added Lysyl endopeptidase (Wako), (mol. ratio of Lysylendopeptidase to the BCDF preparate was 1:200). The mixture was allowedto react at 37° C. for 6 hours so that the BCDF was decomposed intofragments. The reaction solution was subjected to HPLC on a reversephase chromatography column, μ Bondo Pack (0.21×30 cm), which was elutedby gradient elution, while increasing the acetonitrile concentrationfrom 0 to 60% in 0.06% aqueous TFA (eluant) solution so that thefragments were eluted separately from each other. Elution peaks asnumbered 1 to 9 in the HPLC were recovered. Each of the eluates whichcorresponded respectively to the peak No. 1 to 9 was lyophilized, andthe lyophilization product was introduced (incorporated) into proteinsequencer (See; Applied Biosystem Co., Calif., Model 4704). Amino acidsequence was determined according to the method as described in J. Biol.Chem., 193, 265-275 (1951).

Of the fragments mentioned above, the amino acid sequence could bedetermined for fragment Nos. 3, 8, 2 and 6. The amino acid sequenceswere as follows;

Fragments No. 3

Lys-Glu-Ala-Leu-Ala-Glu

Fragments No. 8

Lys-Leu-X-Ala-Gln-Asn-Gln-Trp-Leu-Gln-Y-Met

Fragment No. 2

Pro-Val-Pro-Pro-Gly-Glu-X-Y-Lys

Fragment No. 6

Asp-Val-Ala-Ala-Pro-X

In the above, X and Y are amino acids which could not be determined. Thefragments Nos. 2 and 6 correspond to the N-terminal amino acid sequencesas described in the Example 1.

EXAMPLE 3

This example describes a method for synthesis of an oligonucleotideencoding the amino acid sequence of the BCDF as obtained in Examples 1and 2.

The oligonucleotide was synthesized by a binding reaction of nucleotidesin a phosphorous acid triester method, with silica gel as solid carrier,using DNA synthesizer model 380 A (Applied Biosystem Co., Calif.). Afterremoval of protecting groups in a conventional manner, the deprotectionproduct was subjected to HPLC on a reverse phase chromatography column,Synchropak RP-P (C18) which was eluted by gradient elution whileincreasing acetonitrile concentration, so that the desiredoligonucleotides were obtained in a purified form.

    __________________________________________________________________________                                  Probe No.                                       __________________________________________________________________________    Fragment No. 3                                                                LysGluAlaLeuAlaGlu                                                             ##STR1##                . . .                                                                               3-1                                             ##STR2##                . . .                                                                               3-2                                             ##STR3##                . . .                                                                               3-3                                             ##STR4##                . . .                                                                               3-4                                             ##STR5##                . . .                                                                               3-5                                             ##STR6##                . . .                                                                               3-6                                            (each of 64 types of mixtures)                                                __________________________________________________________________________    Fragment No. 8                                                                LysLeuXAlaGlnAsnGlnTrpLeuGlnYMet                                               ##STR7##                     8-1                                              ##STR8##                     8-2                                             (each of 32 types of mixtures)                                                __________________________________________________________________________    N-terminal amino acid sequence                                                 ##STR9##                                                                      ##STR10##              . . . N-1                                              ##STR11##              . . . N-2                                              ##STR12##              . . . N-3                                             (each of 32 types of mixtures)                                                 ##STR13##               . . .                                                                               N-4                                             ##STR14##               . . .                                                                               N-5                                             ##STR15##              . . . N-6                                              ##STR16##               . . .                                                                               N-7                                            (each of 64 types of mixtures                                                 __________________________________________________________________________

EXAMPLE 4

(1) RPMI 1640 medium (1 l) (glutamine (2 mM), 2 ME (5×10⁻⁵ M),penicillin (100 units/ml), streptomycin (100 μg/ml), gentamycin (20μg/ml), NaHCO₃ (16 mM)) which further contains 20% FCS was placed in a2l-plastic roller type fermentor (roller culture bottles) (Falcon #3027)(hereinafter referred to as roller). Cells (2×10⁵ /ml) of VT-1 wereinoculated in the medium in the roller and cultured at 8 rpm and at 37°C. for 3 days. After the cultivation, the cultured mixture wascentrifuged to collect the cells which were washed twice with PBS. Thecells (1.8×10⁹ /ml) were then suspended in PBS solution (800 ml) andwashed by centrifugation. This procedure was repeated twice to wash thecells. The cells were suspended in RSB solution (Tris-HCl (pH 7.5) (10mM), NaCl (10 mM), MgCl₂ (1.5 mM)) (800 ml) which further contained anuclease inhibitor; ribonucleosides-vanadyl complex (10 mM). Thesuspension thus obtained was admixed with NP-40 (0.05%) and stirredslowly. The mixture was centrifuged at 3000 rpm for 5 minutes toprecipitate cell debris, containing nuclei, which was then removed. Theresultant supernatant was admixed with SDS (final concentration: 0.5%)and EDTA (final concentration: 5 mM), followed by treatment with equalvolume of phenol so that cytoplasmic RNA was recovered by extraction.The extraction with phenol was repeated three times and the extractsthus obtained were combined and admixed with a two-fold volume ofethanol to deposit a precipitate of RNA which was recovered bycentrifugation and was dissolved in Tris-HCl (10 mM) (pH 7.5). RNA wasrecovered from VT-1 cells in a yield of 30 mg.

mRNA was obtained from the RNA in the following manner.

The RNA obtained was subjected to chromatography on a column filled witholigo (dT)-cellulose (P.L. Biochemicals, Type 7). The RNA dissolved in asolution containing Tris-HCl (20 mM) (pH 7.5), NaCl (0.5M), EDTA (1 mM),and SDS solution (0.5%) was passed through the column, prepared as aboveto effect the absorption, and which was washed with a buffer solution(Tris-HCl (20 mM) (pH 7.5), NaCl (0.5 M) EDTA (1 mM)) and then elutedalternately with water and Tris-HCl (10 mM) (pH 7.5), so that mRNA waseluted out. The yield of mRNA thus eluted was 576 μg.

(2) From the mRNA (5 μg) as prepared in the step (1) above,double-stranded cDNA was prepared as follows:

Double-stranded cDNA was prepared according to the Amersham protocolusing a cDNA-synthesizing kit (Amersham) according to the methoddescribed in GUBLER, U and HOFFMAN, B, J., (Gene 25, 263, 1983). Thus,reverse transcriptase was reacted on mRNA to synthetize single-strandedcDNA. Escherichia coli ribonuclease H was made to act on hybrid of mRNAand cDNA as substrate to form nicks and gaps in the RPMI linkage. mRNAwas replaced by the DNA by nick-translation type of reaction usingEscherichia coli DNA polymerase I so that double-stranded DNA wasprepared. The double-stranded DNA was modified by removing the smallover-hang at the 3'-terminal sequence by the use of T4 DNA polymerase.

The double-stranded cDNA was finally obtained in a yield of 1.08 μg.

(3) The double-stranded cDNA (1.08 μg) thus obtained was fractionated bysucrose density gradient centrifugation (from 5 to 25% sucrose densitygradient in a solution (pH 7.5) containing Tris-HCl (50 mM) EDTA (1 mM),as centrifuged at 40000 rpm for 13 hours at 4° C.). Some fractions thusfractionated were analyzed on autoradiogram prepared by agarose gelelectrophoresis. The fractions corresponding to the double-stranded cDNAhaving a size of above 500 bp were combined and admixed with ethanol torecover the double-stranded cDNA (about 0.6 μg) as a precipitate.

(4) A mixture of 0.1M potassium cacodylate (adjusted to pH 7.2 withTris-base), DTT (10 mM), CoCl₂ (2 mM), ³² P-dCTp (0.5 mM) (specificactivity: 1×10⁶ cpm/n mole), double-stranded cDNA (0.6 μg) anddeoxynucleotidyl terminal transferase (BRL) was incubated at 24° C. for20 minutes, follows by treatment with phenol. The mixture was passedthrough a column filled with Sephadex G-50 thereby fractions of cDNAwere collected. The fractions of cDNA thus collected were combined andwas admixed with ethanol to deposit the dC-tailed cDNA (0.24 μg). In amolecule of the cDNA, about 13 of dCMP residues are combined to the3'-terminal sequences of the both sides.

(5) cDNA expressing vector pQ which functions in monkey cells (COScells) was constructed from pCEIL-2 (See: Nature, 302, 305 (1983)) asshown in FIG. 1. pQ vector can insert cDNA to the promoters from eitherside to express peptide protein to be encoded by the cDNA in COS cells.The pQ vector can also be replicated in the cells of Escherichia coli,and can be selected as resistant cells against tetracycline.

The pQ vector was cleaved with Pst I, to which was combined about 13 dGtails in the same manner that dC tails were combined to the 3'-terminalon both sides of a ds-cDNA.

The dG-tailed pQ (100 ng) was mixed with dC-tailed ds-cDNA (20 ng) in anaqueous solution containing Tris-HCl (50 mM) (pH 7.5), NaCl (0.1M), EDTA(1 mM) and the mixture was incubated successively at 65° C. for 2minutes, at 45° C. for 60 minutes, at 37° C. for 60 minutes and finallyat room temperature for 60 minutes. The annealed DNA products thusobtained were introduced into competent cells of E. coli MC 1061.Preparation of competent cells of E. coli MC 1061 and introduction ofthe annealed DNA into the cells prepared were performed in the followingmanner.

Cells of E. coli MC 1061 were inoculated into Ψ medium (100 ml) (2%trypton, 0.5% yeast extract, 0.5% MgSO₄.7H₂ O, pH 7.6) and cultured at37° C. under stirring until the absorbance (optical density) at 550 nmof the culture solution became around 0.3 to 0.5. After the completionof the cultivation, the cultured solution was allowed to stand at 0° C.for 5 minutes, followed by centrifugation to harvest the cells whichwere then suspended in the TfbI (40 ml) (potassium acetate (30 mM),RbCl) (100 mM), CaCl₂ (10 mM), MnCl₂ (50 mM), glycerine (15%), pH 5.8)and the suspension was allowed to stand at 0° C. for 5 minutes.

The suspension was again centrifuged to harvest the cells which werethen suspended in the TfbII (40 ml) (10 mM MOPS or PIPES, CaCl₂ (75 mM),RbCl (10 mM), glycerine (10%), pH 6.5), followed by standing at 0° C.for 15 minutes. The suspension thus treated was divided into portions,which were allowed to stand at -70° C.

Competent cells of E. coli MC 1061 (100 μl), prepared as describedabove, were allowed to stand at 0° C. for 15 minutes, to which wereadded the annealed product (10 μl) of dG-tailed pQ vector with dC-tailedcDNA and an aqueous solution (90 μl) containing MgCl₂ (50 mM) and CaCl₂(10 mM), and the resultant mixture was allowed to stand at 0° C. for 20minutes. The mixture solution was heated to 37° C. for 60 seconds andthen kept at 0° C. for 1˜2 minutes, to which was added Ψ medium (1 ml).The mixture solution was cultured at 37° C. under stirring for 60minutes.

The cultured solution thus obtained was spread on an agar plate of Lbroth (1% trypton, 0.5% yeast extract, 0.5% NaCl, 0.1% glucose) whichfurther contains tetracycline (15 μg/ml) and streptomycin (25 μg/ml) andincubated at 37° C. overnight, to form colonies thereon.

(6) The cloned transformant strains (about 150,000 strains) weresubjected to colony hybridization using probes 8-1 and 8-2, according tothe method described in "Grunstein, M. et al; Methods in Enzymology, 68,379 (1979)". As the result, there were detected 10 cloned strains whichhybridize with probe 8-1. The cloned strains thus obtained were againsubjected to colony hybrization using probes from 3-1 to 3-6, in thesame manner as described above, so that one cloned strain was found tohybridize with the probe 3-2. Plasmid DNA, which the cloned straincontained, was isolated and purified in a conventional manner. Theplasmid DNA was cleaved with restriction enzyme Pst I and the cleavageproduct was subjected to agarose electrophoresis; thereby the cDNAinsert was separated from pQ vector.

The plasmid DNA was subjected to Southern hybridization using probes8-1, 8-2, 3-1→3-6 and N-1→N-7. As the result, it was found that theplasmid DNA hybridizes with 8-1, 3-2, N-2 and N-5, but does nothybridize with any other probes. This plasmid DNA was named pBSF 2-38.It is also apparent that the cDNA insert, which the plasmid DNA (pBSF2-38) contains therein has nucleotide sequences corresponding to some ofthe partial amino acid sequences detected in Example 2. The cDNA hasthus been identified as a gene encoding BCDF.

EXAMPLE 5

(1) Preparation in a large quantity of plasmid DNA (pBSF 2-38):

Cells of E. coli MC 1061 strain (FERMBP-1402) containing the pBSF 2-38were inoculated into Ψ medium containing tetracycline (20 μg/ml) andstreptomycin (25 μg/ml) and cultured at 37° C. with shaking for 5 to 7hours. To the cultured solution was further added freshly prepared Ψmedium (100 ml) containing chloramphenicol at 170 μg/ml as the finalconcentration, and cultured, with shaking, overnight.

Plasmid DNA thus amplified was purified as follows. The culturedsolution obtained as above was centrifuged to harvest the cells whichwere suspended in a buffer solution (5 ml) (50 mM Tris-HCl, pH 7.5). Thesuspension was frozen at -80° C., followed by thawing. The suspension,after thawing, was admixed with lysozyme added (1 mg/l as finalconcentration) and the mixture was allowed to stand at 0° C. for 10minutes. To the mixture was further added EDTA at 0.1M as finalconcentration, and allowed to stand at 0° C. for 10 minutes. The mixturewas then admixed with Triton X-100 at 0.1% as final concentration, andallowed to stand at 0° C. for 60 minutes. The mixture was centrifuged at30,000 rpm for 30 minutes to obtain a supernatant solution. Thesupernatant was admixed with an equal volume of phenol saturated withwater and the aqueous layer separated was again admixed with an equalvolume of chloroform. To the aqueous layer separated was added RNasesolution at 20 μg/ml as final concentration and the mixture solution wasincubated at 37° C. for 60 minutes. The incubated solution was admixedwith 0.2 volume of 5 M NaCl and 1/3 volume of polyethylene glycol andthe mixture solution was then allowed to stand at 0° C. for 60 minutes,followed by centrifugation at 10,000 rpm for 20 minutes to recover DNAas precipitate.

The precipitate recovered was dissolved into water (3.8 ml), to whichwas added CsCl (4 g) and the resultant solution was admixed with EtBr200 μl of 10 mg/ml. The mixture was ultracentrifuged at 40,000 rpm for16 hours at 20° C.

Plasmid DNA fraction recovered, after the ultracentrifugation wasextracted 4 times with 1 to 2 volumes of n-butanol saturated with waterto remove EtBr therefrom. Solution of plasmid DNA was dialyzed againstH₂ O to remove CsCl, and the solution was admixed with 1/10 volume of 3Maqueous solution of sodium acetate (pH 5.6) and then with 2 volume ofcold ethanol, and the mixture was allowed to stand at -20° C. overnight,thereby a precipitate was deposited. The mixture was centrifuged torecover the precipitate which was washed with 80% aqueous ethanolsolution and thoroughly dried. The solid matter thus obtained wasdissolved in a buffer solution (10 mM Tris-HCl, pH 7.5) (50 μl) whichwas served as the sample for transfection in monkey cells.

(2) Transfection of plasmid into COS-7 cells of monkey; COS-7 cells(1×10⁵ /ml) were suspended in RPMI containing FBS (fetal bovine serum)(10%). 3 ml portion of the suspension was placed in a shale (6 cm indiameter) and cultured at 37° C. overnight in a CO₂ gas (5%) incubator.Next morning, the culture supernatant was removed, and, to the residue,was again poured a freshly prepared RPMI (3 ml) containing FBS (10%),and then cultured at 37° C. for 2 hours in a CO₂ gas (5%) incubator.After the cultivation, the supernatant was removed and the cell waswashed once with 2.5 ml of TBS (Tris-HCl (25 mM), pH 7.5, NaCl (130 mM),KCl (5 mM), Na₂ HPO₄ (0.6 mM)). The adherent COS-7 cells was admixedwith a mixture of plasmid [TBS (+), namely TBS mixed with 0.7 mM CaCl₂and 0.5 mM MgCl₂ (10 ml); plasmid DNA (2 γ), DEAE-dextran (10 mg/ml) (50μl)] and the mixture was cultured at 37 ° C. for 4 hours in a CO₂ gas(5%) incubator. After removal of the supernatant which appeared duringthe cultivation, the culture was washed with TBS (2.5 ml), to which wasadded RPMI (2.5 ml) containing FBS (10%) and further chloroquine (150μM). The mixture was cultured at 37° C. for 5 hours in a CO₂ gas (5%)incubator. After the removal of the supernatant appeared during thecultivation, the culture was washed with TBS (2.5 ml), to which waspoured RPMI (2.5 ml) containing FBS (10%) and further chloroguine (150μM). The mixture was cultured at 37° C. for 5 hours in a CO₂ gas (5%)incubator. After the removal of the supernatant the culture was washedtwice (each of 2.5 ml) with TBS. The culture was again admixed with theRPMI (3 ml) containing FBS (10%) and cultured at 37° C. overnight in aCO₂ gas (5%) incubator. After the removal of the supernatant, theculture was again admixed with the RPMI (3 ml) containing FBS (10%) andcultured at 37° C. for 2 days in a CO₂ gas (5%) incubator. The culturemixture was centrifuged to recover the supernatant which was used as thesample for the assay of BCDF activity.

The supernatant of COS culture was assayed for BCDF activity by the useof CL4. The results obtained are shown in FIGS. 2 and 3.

As apparent from the data, COS-7 cells to which pBSF 2-38 plasmid DNAwas transfected exhibit BCDF activity higher than the control. FIG. 2shows activity, based on IgM assayed by an ELISA of the supernatant ofmonkey cells (COS-7) to which was transfected pBSF 2-38 cDNA. FIG. 2shows the activity of purified BCDF (1 U/ml) obtained from VT-1, 657- 7,8, 9 in the supernatant of the culture cells (COS-7) to which wasintroduced BCDF-cDNA, and 657-1 is the supernatant of culture cells(COS-7) to which was introduced lymphokine cDNA.

FIG. 3 shows BCDF activity, as determined by reverse-plaque method, ofthe supernatant of monkey culture cells (COS-7) to which was introducedpBSF 2-38 cDNA. In the FIG. 3, o is the BCDF activity of the supernatantof monkey culture cells (COS-7) cultured without any additives. 657-1,657-7, 8, 9 are the same as defined in FIG. 2. BCDF having a potency of50 U/ml is used as standard.

Recombinant human BCDF produced in the cells of eukaryote (COS-7) asdescribed above was purified by passing the culture solution through acolumn for immobilizing antibody of BCDF and by subjecting the eluate toreverse phase HPLC (Synchropak (C18)). The BCDF was found to contain asaccharide moiety which may be attributable to the existence of anN-glycosylation site, from the amino acid sequence encoded in the cDNA.

The BCDF now obtained coincides in its physicochemical properties asshown below with the purified BCDF obtained from the supernatant of theVT-1, culture by the method described in the Example 1:

(i) Molecular weight: 3.5±0.5×10⁴ dalton (assayed by gel-filtration)2.2±0.2×10⁴ dalton (assayed by SDS-polyacrylic amide electrophoresis)

(ii) Isoelectric point: pH 4.9-5.1

EXAMPLE 6

pBSF 2-38 prepared in Example 5, step (1) was cleaved with restrictionenzyme Bam HI, thereby BCDF cDNA insert was obtained. The BCDF cDNAinsert thus obtained was used as probe for the assessment of BCDF mRNA.mRNA to be used was prepared from various origins such as VT-1 cellscapable of the BCDF concerned; CESS, RPMI 1788 which are estimated toproduce BCDF; tonsilar cells stimulated with TPA; CL4, Jurkat and CEM inwhich BCDF activity is not detected, and human tonsilar cells which wasnot stimulated, in the same manner as in the Example 4, step (1).

A mixture of each mRNA (10 μg/3.6 μl), 5×MOPS buffer solution (0.1M MOPS(pH 7.0), 75 mM NaOAc, 5 mM EDTA) (6.0 μl), formaldehyde (5.4 μl) andformamide (15.0 μl) was incubated at 60° C. for 15 minutes. To themixture was added 3 μl of a coloring solution (80% aqueous glycerolcontaining 0.5% bromophenol blue and 0.05% xylene cyanol), which wasused as the sample to be tested. The sample thus prepared was subjectedto electrophoresis in agarose gel (1.6%) containing 1.8% formaldehydeusing 1×MOPS buffer solution. Blotting onto nitrocellulose filter wasperformed in a conventional manner. The filter was baked at 80° C. for 3hours. The filter thus prepared was dipped in 3×SSC containing 0.1% SDSand prehybridized in a 50 mM sodium phosphate buffer (pH 6.5) containing1×Denhardt solution, 50% formaldehyde, 5×SSC and 250 μg/ml herring DNA,at 42° C. overnight. Hybridization was conducted in a 50 mM sodiumphosphate buffer (pH 6.5) containing 1×Denhardt solution, formamide(50%), 5×SSC and herring DNA (250 μg/ml) using ³² P-labelled pBSF 2-38Bam HI cDNA insert as probe at 42° C. overnight.

The filter thus hybridized was washed 4 times for 5 minutes with 2×SSCcontaining 0.2% SDS at room temperature, and twice for 30 minutes with0.1×SSC containing 0.2% SDS at 50° C. The filter was air-dried and thenwas subjected to autoradiography. Autoradiograms were thus prepared. Theresults obtained show that mRNA derived from VT-1, CESS (BCDF-producingstrain), RPMI 1788 was hybridized with pBSF 2-38 cDNA probe, but mRNAderived from CL-4, Jurkat, CEM and CESS which are not capable ofproducing BCDF was not hybridized with pBSF 2-38 cDNA probe.

mRNA capable of hybridizing with pBSF 2-38 cDNA probe was calculated tobe 15→16S in size.

FIG. 4 shows a part of the same.

Thus, FIG. 4 is an autoradiogram taken by Northern blotting technique,wherein (a) is mRNA of VT-1 cells, (b) is mRNA of T-lymphocytes whichwere prepared by activating human tonsil cells (5×10⁶ ml) with PHA 0.1%,TPA 5 ng for 40 hours, and (c) is mRNA of human tonsil cells which werenot subjected to stimulation treatment.

EXAMPLE 7

MC1061 containing pBSF 2-38 was treated in the same manner as in theExample 5, step (1) to obtain plasmid DNA which was cleaved withrestriction enzyme Bam HI. BCDF cDNA was thus prepared. Restrictionenzyme cleavage map and nucleotide sequence of the BCDF cDNA wereexamined. The nucleotide sequence was determined by the chemical methodof Maxam-Gilbert (See; Meth. Enzym. 65, 499 (1980)) and bydideoxynucleotide chain terminating method (See; F. Sanger et al., Proc.Natl. Acad. Sci. U.S.A. 74, 5463 (1977)) using M13 phage (See; J.Messing et. al., Gene, 19, 269 (1982)). The nucleotide sequence andamino acid sequence thus determined are as shown in FIG. 5. Therestriction enzyme cleavage map is as shown in FIG. 6.

The nucleotide sequence of human BCDF now determined, contains the samepartial structure of the amino acid sequence as disclosed in Examples 1and 2.

EXAMPLE 8

(1) Vector to be used for expressing human BCDF genes was constructuctedas follows: (See: FIG. 7)

DNA fragments (a)→(l) containing the nucleotide sequence shown in FIG. 8were synthesized by the solid phase phosphoric acid triester method. TheDNA fragments, except (a) and (g), were treated with T4 polynucleotidekinase and ATP to phosphorylate the 5'-terminal.

DNA fragments (a)→(l) were combined and annealed, followed by treatmentwith T4 DNA ligase, so that double-stranded synthetic DNA (A) wasformed. pT 9-11 (T. Sato et al, J. Biochem. 101, 525 (1987)) was cleavedwith restriction enzymes HpaI and XbaI and the cleavage product wassubjected to agarose gel electrophoresis, so that large DNA fragment wasisolated. pT 9-11 fragment thus obtained was mixed with synthetic DNA(A) (See; FIG. 8) and ligated by the use of T4 DNA ligase. RecombinantDNA thus obtained ws introduced into the cells of Escherichia coli HB101 strain, from which was selected ampicillin resistant strain. Plasmidobtained from the selected strain was cleaved with restriction enzymes,and the restriction enzyme map was examined. As the result, a strainwhich has pT13S(Nco) involving synthetic HIL-2cDNA was selected.

(2) Recombinant DNA which expresses human BCDF was constructed by theuse of plasmid pT13S(Nco) and pBSF 2-38 (See FIG. 9 and 10):

(i) Plasmid pT13S(Nco) was cleaved with restriction enzymes NcoI andXbaI, and the cleavage products were subjected to agarose gelelectrophoresis, so that larger DNA fragments were isolated in apurified form. Plasmid pBSF 2-38 was cleaved with restriction enzymeBamHI and the cleavage products were subjected to agarose gelelectrophoresis, thereby smaller DNA fragments containing human BCDFcDNA insert were recovered. The human BCDF cDNA insert obtained throughcleavage by the use of restriction enzyme BamHI as above was thencleaved completely by restriction enzyme Xba I, followed by partialcleavage by the use of restriction enzyme MvaI.

Next, a DNA mixture consisting of pT13S(Nco) fragment containingtryptophan promoter and operator (trp p/o) and MvaI-XbaI fragment fromhuman BCDF cDNA was further combined with synthetic DNA(B) [^(5')CATGCCAGTACCACC^(3') and 5'-terminal phosphorylated ^(5')TGGTGGTACTGG^(3') ]. The resultant mixture was ligated by the use of T4DNA ligase. The recombinant DNA thus obtained was introduced into thecells of Escherichia coli HB101, from which strains resistant againstampicillin were selected. The strains thus selected were screened bycolony hybridization to select the strains having the DNA hybridizingwith synthetic DNA (B). From the strains thus finally selected, wasisolated plasmid DNA. The plasmid DNA was subjected to cleavage testwith restriction enzymes and examined for the nucleotide sequence at thebinding site. As the result, the strain containing pTBCDF-01 wasselected. This strain was named pTBCDF-01/HB101.

(ii) Plasmid pBSF 2-38 was subjected to cleavage with restriction enzymeBan I, treatment with DNA polymerase I (Klenow), cleavage withrestriction enzyme XbaI and agarose gel electrophoresis, so that DNAfragments having about 150 base pairs were isolated.

(iii) pTBCDF-01 obtained in (i) above was subjected to cleavage withrestriction enzyme Bam HI, treatment with DNA polymerase I (Klenow),cleavage with restriction enzyme XbaI and agarose gel electrophoresis,so that larger DNA fragments were recovered.

(iv) Two DNA fragments obtained (ii) and (iii) respectively were ligatedby the use of T4 DNA ligase. The resultant recombinant DNA wasintroduced into the cells of Escherichia coli HB101, which were screenedto select the strains resistant against ampicillin. From the strainsthus selected, was isolated plasmid DNA which was subjected to cleavagetest with restriction enzymes, to obtain the strain containing pTBCDF-2.This strain was named pTBCDF-02/Hb101, (FERM P-9061, FERM BP-1403).

(v) Escherichia coli HB101 strain containing plasmid pTBCDF-01 orpTBCDF--02 was cultured in L--broth (1% bactotrypton, 0.5% yeastextract, 0.5% NaCl, 0.1% glucose, pH 7.5) (10 ml) which further containsstreptomycin (25 μg/ml) and ampicillin (25 μg/ml), at 37° C. overnight.5 ml portion of the cultured suspension was inoculated in M9-casaminoacid medium (0.6% Na₂ HPO₄.12H₂ O, 0.3% KH₂ PO₄, 0.05% NaCl, 0.1% NH₄Cl, 0.05% MgSO₄.7H₂ O, 0.00147% CaCl₂, 0.2% glucose, 0.2% casamino acid,0.02% L-leucine, 0.02% L-proline, 0.0002% thiamine hydrochloride, 100μg/ml ampicillin, 25 μg/ml streptomycin, pH 7.4) and cultured at 28° C.for 3 hours. Thereafter, to the cultured mixture was added 3-indoleacetate (IAA) (25 μg/ml), and the culture mixture was further culturedat 23° C. for 21 hours. The culture mixture was centrifuged to harvestthe cells which was washed with 20 mM Tris-HCl buffer (pH 7.5) and NaCl(30 mM) and then was suspended in the buffer of the same composition (8ml). The cells thus obtained was digested with (1%) sodium dodecylsulfate (SDS) or (1 mg/ml) lysozyme in the presence of EDTA (50 mM),followed by sonication (50 W, 30 seconds), so that the proteins in thecells were isolated.

As shown in Table 1, both the isolated solution as prepared from theculture of the strain which has pTBCDF-01/HB101, which has a partialdefect on the side of the 3'-terminus and to this defect is linked apart of human IL-2 cDNA, and the culture of the strain which haspTBCDF-02/HB101 containing BCDF cDNA coding whole human mature BCDFprotein, showed BCDF activity as shown below:

                  TABLE 1                                                         ______________________________________                                        BCDF activity (as determined by Reverse-                                      Plaque method) of the extract prepared from                                   the culture of the strain containing                                          recombinant DNA                                                               Recombinant DNA (strain)                                                                        BCDF activity (U/ml)                                        ______________________________________                                        pTBCDF-01/HB101     800                                                       pTBCDF-02/HB101   28,000                                                      ______________________________________                                    

The recombinant BCDF was purified in the same manner as in the Example1, and then was subjected to HPLC using Synchropak RP-P (C18). A singleprotein was eluted out at the acetonitrile concentration of 50→55%. Onthe protein thus isolated was examined the amino acid sequence in thesame manner as in Example 1, thereby confirming the structure of theN-terminal sequence.

Thus, the polypeptide produced by pTBCDF-02/HB101 has the followingamino acid sequence:

    __________________________________________________________________________    PRO VAL PRO PRO GLY GLU ASP SER LYS ASP VAL                                   ALA ALA PRO HIS ARG GLN PRO LEU THR SER SER                                   GLU ARG ILE ASP LYS GLN ILE ARG TYR ILE LEU                                   ASP GLY ILE SER ALA LEU ARG LYS GLU THR CYS                                   ASN LYS SER ASN MET CYS GLU SER SER LYS GLU                                   ALA LEU ALA GLU ASN ASN LEU ASN LEU PRO LYS                                   MET ALA GLU LYS ASP GLY CYS PHE GLN SER GLY                                   PHE ASN GLU GLU THR CYS LEU VAL LYS ILE ILE                                   THR GLY LEU LEU GLU PHE GLU VAL TYR LEU GLU                                   TYR LEU GLN ASN ARG PHE GLU SER SER GLU GLU                                   GLN ALA ARG ALA VAL GLN MET SER THR LYS VAL                                   LEU ILE GLN PHE LEU GLN LYS LYS ALA LYS ASN                                   LEU ASP ALS ILE THR THR PRO ASP PRO THR THR                                   ASN ALA SER LEU LEU THR LYS LEU GLN ALA GLN                                   ASN GLN TRP LEU GLN ASP MET THR THR HIS LEU                                   ILE LEU ARG SER PHE LYS GLU PHE LEU GLN SER                                   SER LEU ARG ALA LEU ARG GLN MET                                               __________________________________________________________________________

EXAMPLE 9

Construction of recombinant DNA capable of producing conjugated protein(ΔHIL-2-BCDF) of human BCDF with human interleukin-2 (HIL-2) by the useof plasmid pT13S(Nco) (as described in Example 8) and pBSF 2-38. (SeeFIG. 11 and 12):

(i) Plasmid pT13S(Nco) was cleaved with restriction enzyme BglII andXbaI, and the cleavage product was subjected to agarose gelelectrophoresis, so that larger DNA fragment was isolated. Plasmid pBSF2-38 was cleaved with restriction enzyme BamHI, and the cleavage productwas subjected to agarose gel electrophoresis, so that smaller DNAfragment containing human BCDF cDNA insert was isolated. The human BCDFcDNA insert obtained from BamHI cleaved was further treated withrestriction enzyme XbaI, followed by partial cleavage with MvaI. A DNAmixture composed of pT13S(Nco) fragment containing the promotermentioned above and MVaI-XbaI fragment containing human BCDF cDNA wascombined with synthetic DNA(C)[^(5') GATCTCTTCAGAGCCCCAGTACCCCC^(3') ].The mixture was ligated by the use of T4 DNA ligase.

The recombinant DNA thus obtained was introduced into the cells ofEscherichia coli HB101, so that the strains resistant against ampicillinwere selected. The strains were screened by colony hybridization toselect the strain having DNA capably of hybridizing with syntheticDNA(C). Plasmid obtained from the strains selected as above was cleavedwith restriction enzyme. On the plasmid, the nucleotide sequence ofjoining sites was examined. The strain containing pTBCDF-11 was thusobtained, which was named pTBCDF-11/HB101.

(ii) Plasmid pBSF 2-38 was cleaved with restriction enzyme Ban I, andthe cleavage product was subjected to treatment with DNA polymerase I(Klenow), cleavage with XbaI, and agarose gel electrophoresis, so thatthe DNA fragment of about 150 base pairs was isolated.

(iii) pTBCDF-11 obtained in (i) above was cleaved with restrictionenzyme BamHI, and the cleavage product was subjected to treatment withDNA polymerase I (Klenow), cleavage with XbaI and agarose gelelectrophoresis, so that larger DNA fragment was recovered.

(iv) The two DNA fragments obtained in (ii) and (iii) respectively wereligated by the use of T4 DNA ligase. The recombinant DNA thus obtainedwas introduced into the cells of Escherichia coli HB101, from which thestrains resistant against ampicillin were selected. From the strainsthus selected was isolated plasmid DNA which was subjected to cleavagetest with restriction enzymes. The strain containing pTBCDF-12 was thusselected, which was named pTBCDF-12/HB101/(FERM-P9062, FERM BP-1404).

(v) The strains of HB101, namely the HB101 containing plasmid pTBCDF-11wherein the 5'-terminal of the BCDF cDNA is linked to a part of humanIL-2 cDNA and a defect on the side of the 3'-terminus is linked to humanIL-2 cDNA; and the HB101 containing plasmid pTBCDF-12 where the5'-terminus of the whole BCDF cDNA covering mature BCDF protein islinked to DNA responsible to N-terminus amino acid protein in human IL-2cDNA were cultured in the same manner as in Example 8. The culture wasisolated in a conventional manner. The culture solution isolated fromthe two kinds of HB101 strains exhibit BCDF activity as demonstrated inTable 2 below.

                  TABLE 2                                                         ______________________________________                                        BCDF activity (as determined by Reverse-                                      Plaque method) of the culture solution                                        isolated from the culture of the strains                                      containing recombitant DNA                                                    Recombinant DNA (strain)                                                                        BCDF activity (U/ml)                                        ______________________________________                                        pTBCDF-11/HB101      500                                                      pTBCDF-12/HB101   >25,600                                                     ______________________________________                                    

EXAMPLE 10

Cells of pTBCDF-12/HB101 (See Example 9) were cultured according toExample 8. The culture was processed in the following manner to isolateinclusion bodies formed in the cells.

Thus, the culture was centrifuged to collect the cells which weresuspended at a 10-fold density of cells (as compared with the culture)in 20 mM Tris-HCl buffer (pH 7.5) containing 30 mM NaCl, to which wereadded lysozyme (1 mg/ml) and EDTA (0.05M), followed by stirring. Thesuspension was allowed to stand under ice-cooling for 1 hour, and wassubjected to sonication to break (degrade) the cells. The suspensionthus treated was centrifuged at 10000 rpm for 5 minutes, thereby theinclusion bodies were recovered.

The inclusion bodies were solubilized with the addition of 6M guanidinehydrochloride and the solution was adjusted to the final concentrationof 100 μg/ml

of ΔHIL-2-BCDF and 2M of guanidine hydrochloride. The suspension wasadmixed with oxidized glutathione (1 mM) and reduced glutathione (10mM), adjusted to pH 8.0 and allowed to stand at room temperature for 10to 16 hours. The suspension was then subjected to gel filtration(Sephadex G-25), so that guanidine hydrochloride was removed, and thefraction corresponding to ΔHIL-2-BCDF as a buffer solution forKallikrein reaction. The fraction was subjected to SDS-polyacrylamidegel electrophoresis.

Molecular weight now determined by the electrophoresis nearly coincidedwith the value calculated based on its amino acid composition.

Examination of the N-terminal amino acid sequence by means of a proteinsequencer showed that it corresponds to that of HIL-2.

The polypeptide having the BCDF activity is represented by the followingamino acid sequence:

    __________________________________________________________________________    ALA PRO THR SER SER SER THR LYS LYS THR GLN                                   LEU GLN LEU GLU HIS LEU LEU LEU ASP LEU PHE                                   ARG ALA PRO VAL PRO PRO GLY GLU ASP SER LYS                                   ASP VAL ALA ALA PRO HIS ARG GLN PRO LEU THR                                   SER SER GLU ARG ILE ASP LYS GLN ILE ARG TYR                                   ILE LEU ASP GLY ILE SER ALA LEU ARG LYS GLU                                   THR CYS ASN LYS SER ASN MET CYS GLU SER SER                                   LYS GLU ALA LEU ALA GLU ASN ASN LEU ASN LEU                                   PRO LYS MET ALA GLU LYS ASP GLY CYS PHE GLN                                   SER GLY PHE ASN GLU GLU THR CYS LEU VAL LYS                                   ILE ILE THR GLY LEU LEU GLU PHE GLU VAL TYR                                   LEU GLU TYR LEU GLN ASN ARG PHE GLU SER SER                                   GLU GLU GLN ALA ARG ALA VAL GLN MET SER THR                                   LYS VAL LEU ILE GLN PHE LEU GLN LYS LYS ALA                                   LYS ASN LEU ASP ALA ILE THR THR PRO ASP PRO                                   THR THR ASN ALA SER LEU LEU THR LYS LEU GLN                                   ALA GLN ASN GLN TRP LEU GLN ASP MET THR THR                                   HIS LEU ILE LEU ARG SER PHE LYS GLU PHE LEU                                   GLN SER SER LEU ARG ALA LEU ARG GLN MET                                       __________________________________________________________________________

(2) Cleavage with Kallikrein

ΔHIL-2-BCDF (80 μg) as obtained above was reacted with human plasmakallikrein (73.5 μg ) in 50 mM Tris -HCl buffer solution (pH 7.8)containing NaCl (113 mM) at 37° C. for 16 hours. The reaction mixturewas subjected to reverse phase HPLC, thereby the fractions correspondingto human Ala-BCDF as eluted at the concentration of about 55% withrespect to acetonitrile and 0.1% with respect to TFA were collected. Onthe fraction thus obtained the amino acid sequence at the N-terminal wasexamined by the use of a protein sequencer. As the result, it wasconfirmed that ΔHIL-2-BCDF had been transferred (converted)quantitatively into human Ala-BCDF protein. Human Ala-BCDF was recoveredin a yield of 18.03 mg (recovery: 84%). "Human Ala-BCDF" is referred toas a natural human BCDF in which the N-terminal is linked to one alaninemolecule and which has the following amino acid sequence:

    __________________________________________________________________________    ALA PRO VAL PRO PRO GLY GLU ASP SER LYS ASP                                   VAL ALA ALA PRO HIS ARG GLN PRO LEU THR SER                                   SER GLU ARG ILE ASP LYS GLN ILE ARG TYR ILE                                   LEU ASP GLY ILE SER ALA LEU ARG LYS GLU THR                                   CYS ASN LYS SER ASN MET CYS GLU SER SER LYS                                   GLU ALA LEU ALA GLU ASN ASN LEU ASN LEU PRO                                   LYS MET ALA GLU LYS ASP GLY CYS PHE GLN SER                                   GLY PHE ASN GLU GLU THR CYS LEU VAL LYS ILE                                   ILE THR GLY LEU LEU GLU PHE GLU VAL TYR LEU                                   GLU TYR LEU GLN ASN ARG PHE GLU SER SER GLU                                   GLU GLN ALA ARG ALA VAL GLN MET SER THR LYS                                   VAL LEU ILE GLN PHE LEU GLN LYS LYS ALA LYS                                   ASN LEU ASP ALA ILE THR THR PRO ASP PRO THR                                   THR ASN ALA SER LEU LEU THR LYS LEU GLN ALA                                   GLN ASN GLN TRP LEU GLN ASP MET THR THR HIS                                   LEU ILE LEU ARG SER PHE LYS GLU PHE LEU GLN                                   SER SER LEU ARG ALA LEU ARG GLN MET                                           __________________________________________________________________________

(3) Removal of the N-terminal Ala by the use of amino peptidase P

Amino peptidase P to be used for this purpose was purified according tothe method as described in "Methods Enzymol. 19, 521 (1970)".

A solution of human Ala-BCDF as obtained in (2) above was subjected togel filtration on a column of Sephadex G-25 which had been equilibratedwith (50 mM) Tris-HCl buffer solution (pH 8.0) containing MnCl₂ (0.4mM), so that Ala-BCDF fractions were obtained. The fraction of Ala-BCDF(50 μg) was admixed with amino peptidase P, and the mixture was allowedto react at 37° C. for 16 hours. After the reaction, the reactionmixture was subjected to reverse phase HPLC, so that the fractionscorresponding to BCDF were collected. The N-terminal amino sequence ofthe fraction thus obtained was examined by the use of a proteinsequencer. As a result, it was confirmed that the Ala-BCDF had beenconverted quantitatively into BCDF. Table 3 below shows the activity ofAla-BCDF and BCDF.

                  TABLE 3                                                         ______________________________________                                        BCDF activity as exhibited by                                                 pTBCDF-12/HB101 (Reverse-plaque method)                                       Protein produced                                                                           BCDF activity (U/μg protein)                                  ______________________________________                                        Ala-BCDF     20,000                                                           BCDF         20,000                                                           ______________________________________                                    

EXAMPLE 11

Construction of the recombinant DNA, capable of expressing human BCDF,lacking the C-terminus was prepared by the use of plasmids pTBCDF-1 andpT9-11. (See; FIG. 13):

(i) Plasmid pTBCDF-01 was cleaved with restriction enzyme XbaI, thecleavage product being subjected to DNA polymerase I (Klenow) treatment,cleavage with PvuI, and agarose gel electrophoresis, so that smaller DNAfragment containing trp P/O and human BCDF cDNA lacking the 3'-terminus.Plasmid pT9-11 was cleaved with restriction enzyme Bam HI and PvuI, andthe cleavage product was subjected to agarose gel electrophoresis, sothat larger DNA fragment containing trpA terminator was recovered.

The two kinds of DNA fragments thus prepared were mixed together withsynthetic DNA (c) [^(5') CCTAGCCCGGGTGAATGAAT^(3') and5'GATCATTCATTCACCCGGGCTAGG^(3') ], and the mixture was ligated by theuse of T4 DNA ligase. The recombinant DNA thus prepared was introducedinto the ampicillin cells of Escherichi coli HB101, so that ampicillinresistant strain was selected. From the strain was isolated plasmid DNAwhich was then cleaved with restriction enzyme. On the cleavage productwas examined the nucleotide sequence around the linking site, so thatthe strain containing pTBCDF-03 was selected. The strain was namedpTBCDF-03/HB101. The plasmid pTBCDF-03 contains a gene which lacks apart of the 3'-terminal of BCDF cDNA. The polypeptide as isolated frompTBCDF-03/HB101 has the following amino acid sequence;

    __________________________________________________________________________    PRO VAL PRO PRO GLY GLU ASP SER LYS ASP VAL                                   ALA ALA PRO HIS ARG GLN PRO LEU THR SER SER                                   GLU ARG ILE ASP LYS GLN ILE ARG TYR ILE LEU                                   ASP GLY ILE SER ALA LEU ARG LYS GLU THR CYS                                   ASN LYS SER ASN MET CYS GLU SER SER LYS GLU                                   ALA LEU ALA GLU ASN ASN LEU ASN LEU PRO LYS                                   MET ALA GLU LYS ASP GLY CYS PHE GLN SER GLY                                   PHE ASN GLU GLU THR CYS LEU VAL LYS ILE ILE                                   THR GLY LEU LEU GLU PHE GLU VAL TYR LEU GLU                                   TYR LEU GLN ASN ARG PHE GLU SER SER GLU GLU                                   GLN ALA ARG ALA VAL GLN MET SER THR LYS VAL                                   LEU ILE GLN PHE LEU GLN LYS LYS ALA LYS ASN                                   LEU ALA                                                                       __________________________________________________________________________

(ii) The pTBCDF-03/HB101 was treated in the same manner as in Example 8to prepare an isolated culture solution. As shown in Table 4, theculture solution of the pTBCDF-03/HB101 shows BCDF activity.

                  TABLE 4                                                         ______________________________________                                        BCDF activity of the culture solution                                         isolated from the strain containing                                           recombinant DNA (Reverse-plaque method)                                       Strain         BCDF activity (U/ml)                                           ______________________________________                                        pTBCDF-03/HB101                                                                              400                                                            ______________________________________                                    

EXAMPLE 12

The same procedures were applied as in Example 10, step (1) to (3),except that in step (3) Ala-BCDF was used in the quantity of 2.02 mg.

The human BCDF was recovered in a yield of 2.0 mg. Table 5 shows theactivity of human BCDF and of human Ala-BCDF. The activity isrepresented by the unit value as defined according to the method ofProc. Natl. Acad, Sci. USA, 82, 5490 (1985).

                  TABLE 5                                                         ______________________________________                                                     BCDF activity                                                                           Specific activity                                                   (U/ml)    (U/μg)                                              ______________________________________                                        Human Ala-BCDF 3,500,000   5,000                                              Human BCDF     1,500,000   2,500                                              ______________________________________                                    

(4) Formulation of human BCDF

The solution of the fractions containing human BCDF or human Ala-BCDFobtained by HPLC was allowed to stand at -20° C. overnight. The lowerlayer of the solution separated from the upper layer (acetonitrile) wassubjected to gel filtration on Sephadex G-25 or dialysis, the remainingacetonitrile and TFA in the lower layer being removed, so that thesolution was converted into a PBS solution. The solution thus preparedwas diluted and was optionally admixed with (10%) fetal bovine serum(FBS) or with (0.1%) human serum albumin. The for mixture was filteredfor sterile conditions, and finally was fomulated into pharmaceuticallysuitable forms.

EXAMPLE 13

The effect of human BCDF or human Ala-BCDF on the production of specificantibody was examined as follows:

The cells (1×10⁴) of human B cell line SKW6-CL4 were suspended in RPMI1640 medium (100 μl) containing (10%) FBS, to which was added serialdilutions (2-fold) (100 μl) of human BCDF (79 ng/ml) or human Ala-BCDF(57 ng/ml). The suspension was cultured on a plate (96 well) (CorningCorp. 25860) at 37° C. in 5% CO₂ for 3 days. IgM in the supernatant wasassayed according to the ELISA method (See; Proc. Natl. Acad. Sci. USA,82, 5490 (1985).

As shown in the FIG. 14, SKW6-CL4 cells cultured together with humanBCDF or human Ala-BCDF exhibit a significantly elevated production ofantibody (IgM), as compared with the control. There was not observed astatistically significant difference in the antibody production betweenhuman BCDF and human Ala-BCDF.

Human Ala-BCDF of known activity which was prepared by IL-2 fusionprotein method was diluted into the portions (each 100 U/ml) which wasused as BCDF standard.

EXAMPLE 14

Cells of SKW6-CL4 were cultured with human BCDF (1 U/ml) (200 pg/ml) andhuman recombinant IL-2 (50,000 U/ml) 1 μg/ml to 0.32 pg/ml) under thesame condition as in the Example 13, for 3 days.

The number of cells which were differentiated into the cells to produceantibody were enumerated by Reverse PFC method (Eur. J. Immunol, 13, 31(1983)). As shown in FIG. 15, the cells of SKW6-CL4 when cultured in thepresence of human BCDF and human IL-2 added simultaneously exhibit anenhanced production of the antibody, as compared with the cells culturedin the presence of human BCDF or human IL-2 each added singly. The sameresult was also observed in ELISA method.

EXAMPLE 15

Spleen cells of DBA/2 mouse (female, 8 weeks old) were treated with 0.9%NH₄ Cl in a conventional manner to remove erythrocytes therefrom. Thecells were then treated with anti-Thy 1 antibody and guinea pigcomplement to remove T-cells, so that B cells fraction obtained in apurified form.

The B cells fraction thus obtained (7.5×10⁵) and sheep red blood cell(SRBC) (1×10⁵) were suspended in RPMI 1640 medium (200 ul) containing(5%) FBS. The suspension was cultured on a plate (96 well) (CorningCorp. 25860) at 37° C. in 5% CO₂ for 4 to 5 days. During thecultivation, human BCDF (125 U/ml: 50 ng/ml) and human IL-2 (400 U/ml: 8ng/ml) were added suitably to the culture at different intervals.

After the cultivation, the cells capable of producing anti-SRBC antibodywere enumerated by PFC method (The Method of Immunological Experiment,P.479; Jap. Soc. Immunology).

As shown in FIG. 16, human BCDF stimulates the production of specificantibody in spleen cells of the normal mouse. The effect was furtherstrengthened by the combined application of human BCDF and human IL-2.The same effect was observed in other strains of mouse, for example inBalb/c mouse. The test for the spleen cells derived from Balb/c nu/numouse which had lost T-cell function because of the lack of the thymus,gave also the same result as shown in the FIG. 17.

EXAMPLE 16

SRBC (1×10⁸) as antigen was injected intravenously to the tail of DBA/2mouse (female, 8 weeks old), so that the DBA/2 mouse was sensitized.Four days after, the spleen was removed out from the sensitized mouse.The spleen was treated according to the method described in Example 15to prepare sensitized B cells fractions in a purified form. Thesensitized B cells (7.5×10⁵) and antigen SRBC (1×10⁵) as antigen weresuspended in RPMI 1640 medium (200 ml) containing FBS (5%) and thesuspension was cultured at 37° C. in the presence of human BCDF (50ng/ml) and human IL-2 (8 ng/ml to 80 pg/ml) in 5% CO₂ for 4 days. Thecells capable of producing anti-SRBC antibody were enumerated directlyaccording to PFC method.

As shown in FIG. 18, addition of human BCDF causes a statisticallysignificant increase in the production of the specific antibody, ascompared with the control (without the addition of human BCDF). Theeffect of the human BCDF on the productivity of the specific antibodywas further strengthened when the human BCDF was applied together withhuman IL-2.

EXAMPLE 17

(1) Enhancement of production of the antibody against the antigensensitized in vitro

Immuno-suppresive drug, hydrocortisone (HC) (Trade mark of aqueoussuspension of hydrocortisone acetate, Shuroson F, Japan Shering) (5 mg)was injected subcutaneously to Balb/c mouse (female, 6 weeks old), sothat the mouse was immunosuppressed.

1, 2 and 7 days after, the spleen was removed from the immunosuppressedmouse. The spleen was treated with (0.9%) NH₄ Cl to remove red bloodcells, so that purified spleen cells were prepared.

The spleen cells thus prepared (5×10⁵) and SRBC (1×10⁵) were suspendedin RPMI 1640 medium (200 μl) containing FBS (5%), and the suspension wascultured on a plate (96 well) (Corning Corp. 25860) at 37° C. in 5% CO₂.To the culture medium were added human Ala-BCDF (25 U/ml; 4 ng/ml),human IL-2 (400 U/ml; 8 ng/ml) or T cell factor (TF; culture supernatantof hybridoma prepared from T cell stimulated with concanavalin A) (10%)as a positive control. On the 5th day of the cultivation, the cells wererecovered in a conventional manner. The cells producing anti-SRBCantibody were enumerated directly according to PFC method.

As shown in the Table 6, the productivity of the specific antibody inthe mouse where the formation of PFC at the addition of TF had beenreduced by the hydrocortisone applied, as compared with the control, wasenhanced by the addition of human Ala-BCDR and also in combination withhuman IL-2.

                  TABLE 6                                                         ______________________________________                                                                         PFC/1 × 10.sup.6                       Treatment                                                                              SRBC     Lymphokine     Spleen cells                                 ______________________________________                                        HC untreated                                                                           (-)      (-)             0                                           mouse    (+)      (-)             2                                                    (+)      TF             325                                                   (+)      human IL-2     26                                                    (+)      human Ala-BCDF 14                                                    (+)      human IL-2 +   35                                                             human Ala-BCDF                                              One day after                                                                          (+)      (-)             0                                           HC treatment                                                                           (+)      TF             250                                                   (+)      human IL-2      6                                                    (+)      human Ala-BCDF 38                                                    (+)      human IL-2 +   23                                                             human Ala-BCDF                                              4 days after                                                                           (+)      (-)             5                                           HC treatment                                                                           (+)      TF             111                                                   (+)      human IL-2     18                                                    (+)      human Ala-BCDF 54                                                    (+)      human IL-2 +   83                                                             human A±a-BCDF                                                    (+)      (-)            14                                           7 days after                                                                           (+)      TF             152                                          HC treatment                                                                           (+)      human IL-2      6                                                    (+)      human Ala-BCDF  2                                                    (+)      human IL-2 +   38                                                             human Ala-BCDF                                              ______________________________________                                    

(2) Enhancement of production of the antibody against the antigensensitized in vivo

Hydrocortisone (0.5 mg) was injected subcutaneously to Balb/c mouse(female, 6 weeks old), and 7 days after, SRBC (1×10⁸) was injectedintravenously to the tail of the mouse. On the 3 days after theinjection, spleen was removed from the mouse, and the spleen was treatedin the same manner as in (1) above to prepare the spleen cells. Thecells producing specific anti-SRBC antibody were enumerated directlyaccording to the PFC method.

The spleen cells (5×10⁵) was cultured together with SRBC (1×10⁵) in thesame manner as in (1) above. To the culture medium were added human (100U/ml, 20 ng/ml), human IL-2 (400 U/ml, 8 ng/ml) or T-cell factor (10%).On the 4th day of the cultivation, the cells were recovered and thecells producing anti-SRBC antibody were enumerated directly according tothe PFC method.

As shown in the Table 7, by the treatment with hydrocortisone theproduction of antibody against the antigen sensitized in vivo wassuppressed about 60%. The spleen cells thus treated were cultured in thepresence of lymphokine and SRBC for 4 days. The comparison of the spleencells of the mouse treated with hydrocortisone and the spleen cells ofthe mouse without treatment with hydrocortisone shows that thesuppressing effect of hydrocortisone is remarkably reduced (by about20%) in the spleen cells of the mouse treated with human Ala-BCDF. Therewas not observed statistically significant effect of the hydrocortisonein the spleen cells treated either with TF or with human IL-2, ascompared with the control.

The enhanced productivity of the antibody by the use of BCDF in themouse administered with hydrocortisone demonstrates that immunity andhost defense mechanism are effectively activated by the BCDF, underimmunosuppresive condition.

                  TABLE 7                                                         ______________________________________                                                       PFC/1 × 10.sup.6                                                                   Suppression by                                      Treatment      spleen cells                                                                             HC treatment                                        ______________________________________                                        Balb/c SRBC(-)  1                                                             SRBC(+)        104         0%                                                 SRBC(+)         42        60%                                                 HC treatment                                                                  [Lymphokine treatment in vitro PFC]                                                                   PFC/1 ×                                                                            Suppression                                         in vitro       10.sup.6   by HC                                      Treatment                                                                              SRBC    lymphokine spleen cells                                                                           treatment                                ______________________________________                                        HC untreated                                                                           -       -           27                                               SRBC     +       -          290      0                                        administration                                                                         +       TF         750      0                                                 +       human IL-2 133      0                                                 +       human      573      0                                                         Ala-BCDF                                                              +       human IL-2+                                                                              506      0                                                         human                                                                         Ala-BCDF                                                     HC 0.5 mg                                                                              -                  --       0                                        treatment                                                                     SRBC     +       -           83      71                                       administration                                                                         +       TF         276      63                                       SRBC     +       human IL-2  43      67                                       administration                                                                         +       human      443      22                                                        Ala-BCDF                                                              +       human IL-2+                                                                              200      60                                                        human                                                                         Ala-BCDF                                                     ______________________________________                                    

EXAMPLE 18

(Growth of bone marrow cells stimulated by human Ala-BCDF) Hematopoieticstimulating effect of human BCDF and of human Ala-BCDF was examined inthe following procedures;

The thigh bone was removed from DBA/2 mouse (female, 8 weeks old), fromwhich was prepared bone marrow cells under sterile condition in aconventional manner as described in "The Method of ImmunologicalExperiment, P. 1305, Jap. Soc. Immunology". The bone marrow cells(5×10⁴) thus obtained were suspended in RPMI 1640 medium (100 μl)containing 10% FBS, to which was added 100 μl solution composed ofserial dilutions (2 fold) of human Ala-BCDF (500 U/ml: 100 ng/ml), andof mouse IL-3 (5 U/ml, 1.25 U/ml). The suspension was cultured on aplate (96 well) (Corning 25860) at 37° C. in 5,% CO₂, and the effect onthe (growth) of bone marrow cells was examined.

The mouse IL-3 was prepared in the following manner. Thus, plasmid inwhich mouse IL-3 cDNA (See Nature 307, 233 (1984)) had been integratedinto pQ vector (See: Japanese Patent Application No. 184858/1986) wastransfected into COS cells in a conventional manner as described inProc. Natl. Acad. Sci, USA 81, 1070 (1984)) and the COS cells werecultured for 3 days. The supernatant separated from the culture (IL-3activity; about 50 U/ml) was dialyzed against RPMI 1640 medium, whichwas served for the subsequent procedure. On the 4th day of cultivation,the culture of bone marrow cells was admixed with tritium labeledthymidine (3H-TdR) (1 μCi/well) and allowed to stand for 18 hours. Theculture was filtered to collect the cells on which the 3H-TdRincorporated was assayed by means of a β-scintillation counter.

As shown in FIG. 19, the culture of bone marrow cells cultured withhuman Ala-BCDF exhibits an enhanced ³ H-TdR incorporation of the bonemarrow cells, as compared with the control. When human Ala-BCDF wasadded in combination with bone marrow cell growth factor (mouse IL-3),the growth of the bone marrow cells was further enhanced.

The same phenomenon was also observed for other strains of mice, such asBalb/c, c57B1/6 and C3H/HeN.

EXAMPLE 19

Lentinan (10 mg/kg) was injected intravenously into the tail of DAB/2mouse (female, 8 weeks old). 10, 7 and 5 days after the injection, bonemarrow was removed from the mouse, and the bone marrow cells wereprepared according to the method as described in example 18 above.

Bone marrow cells were cultured in the presence of human Ala-BCDF(50U/ml; 10 ng/ml) and mouse IL-3 (5 U/ml). The effect of BCDF on thegrowth of the cells was examined in the culture. Thus, on 3, 4 and 5 thedays of the cultivation, the culture was admixed with 3H-TdR (1μCi/well). The growth of the bone marrow cells were assessed based onthe incorporation of 3H-TdR into the cells according to the methoddescribed in example 18 above.

Thus, lentinan was administered to a female DBA/2 mouse. 10, 7 and 5days after the administration, the DBA/2 mouse treated and the one whichwas not administered with lentinan were treated in the same manner toprepare bone marrow cells. To the bone marrow cells was added BCDF (50ng/ml) and IL-3 (5 U/ml). The rate of growth was assessed on the basisof the incorporation of 3H-TdR on the 3, 4 and 5 day after the additionof BCDF and IL-3. LNT means lentinan.

As shown in FIG. 20, in the bone marrow cells of the mouse administeredwith lentinan 5 or 7 days before the removal of the bone marrow, thegrowth was enhanced by the combined application of human Ala-BCDF andmouse IL-3, as compared with control.

EXAMPLE 20

Bone marrow cells were prepared from DBA/2 mouse (female, 8 weeks old)according to the method as described in example 18 above. Colony formingactivity was determined by the use of the assay system as describedbelow. Thus, DMEM medium as concentrated 2 folds) containing (20%) FBS,(5×10⁵) 2-mercaptoethanol was admixed with (1%) agar solution, heated tomelt agar in the water-bath in a ratio of 7:3. The bone marrow cells(7.5×10⁴) mentioned above was suspended in the aqueous agar medium asprepared above, and the suspension was semi-solidified in a 3.5 cmschale (Falcon 1008). The agar suspension was cultured at 37° C. in 5%CO₂ for 7 days to develop colonies (more than 50 cells) cells andclusters (consisting of 8 to 50 cells). The number of colonies andclusters were enumerated microscopically.

The agar suspension medium was admixed with human Ala-BCDF (50 to 500U/ml; 10 to 100 ng/ml) and mouse IL-3 (1.25 U/ml) and cultured. For thisculture, was examined the formation of colonies and clusters.

As shown in FIG. 21, the agar suspension medium added with humanAla-BCDF exhibits a statistically significant increase in the number ofclusters, as compared with the control. FIG. 21 also shows that thecolony formation induced by mouse IL-3 is further enhanced by theaddition of BCDF.

The same phenomenon was observed in the immunosuppressed mouse inducedby the application of immuno-suppressing agent, hydrocortisone. Thus,hydrocortisone (1 mg to 5 mg) was injected subcutaneously to DBA/2 mousemarrow (female, 6 weeks old). 5 days after, bone marrow cells wereprepared in the same manner as described above.

Human Ala-BCDF (200 ng/ml, 1000 U/ml), mouse IL-3 (0.6 U/ml) were addedto DMEM medium containing (1%) methylcellulose (20%) FBS and (5×10⁵ M)2-methylmercaptoethanol. To the medium thus prepared was added bonemarrow cells (2×10⁴), and cultured for 7 days to develop colonies. Asshown in FIG. 22, the formation of clusters was enhanced significantlyby the addition of human Ala-BCDF. By the administration ofhydrocortisone, the colony-forming ability itself was increased, and theformation of macrophage lymphocyte like colonies was remarkably enhancedby the addition of human Ala-BCDF in combination with mouse IL-3.

Therefore, it was confirmed that BCDF applied either singly or incombination with IL-3 stimulates hematopoietic function inimmunosuppressed mice.

EXAMPLE 21

It was examined whether human BCDF or human Ala-BCDF can differentiatetumor cells and exhibit an antitumor activity.

The test was conducted as follows:

Human myelomonocitic leukemia cell line THP-1 and KG-1 (each 2×10⁵ /ml)was suspended in RPMI 1640 medium containing (10%) FBS. The suspensionof cell were added in an amount of 1 ml/well onto a plate (Corning Co.,25820), and cultured at 37° C. in CO₂ (5%) for 2 days. To the culturewas added human Ala-BCDF (20 ng/ml to 5 pg/ml) and the effect of theaddition of the human Ala-BCDF on the differentiation of the tumor cellswas examined. Thus, the frequency of occurrence of FC receptors in tumorcells on the 2nd day of the culture was determined by rosette formationmethod (See: Cancer Research, 44, 5127 (1984)) using sensitized ox redblood cells. The value of the frequency of occurrence was used as theindex of differentiation. FIG. 23 shows frequency of occurrence of Fcreceptors as measured for the culture of THP-1, KG-1 which were culturedin a medium containing RPMI 1640 (2 10.sup. 5 /ml) and FBS (10%) for 2days. The frequency of occurrence was determined by rosette formationmethod using sensitized ox red blood cells. As shown in FIG. 23, humanAla-BCDF enhanced significantly the frequency of occurrence of Fcreceptors in myelomonocytic leukemia cell lines, THP-1 and KG-1.Therefore it is apparent that BCDF can stimulate the differentiation oftumor cells into normal cells and BCDF exhibits anti-tumor activity.

EXAMPLE 22

Tonsils were obtained from patients who had undergone tonsillectomy.Tonsils were teased in RPMI-1640, after removal of the debris andmononuclear cells were separated by centrifugation on LSM (Ficoll-sodiumdiatrizoate) solution at 400 xG for 30 min.

Tonsil mononuclear cells (1→2×10⁵ /200 μl 5 RPMI 1640+10% FBS/well) werestimulated with several concentrations of PWM (pokeweed mitogen) andvarious concentration of BCDF. After day 7 incubation, the concentrationof IgM, IgG and IgA in culture supernatants were quantitated by asandwich enzyme-linked immunosorbent assay(ELISA).

The result showed that BCDF increased the production of variousantibodies (FIG. 24).

EXAMPLE 23

Tonsils were obtained from patients who had undergone tonsillectomy.Tonsils were teased in RPMI-1640, after removal of the debris, andmononuclear cells were separated by centrifugation on LSM (Ficoll-sodiumdiatrizoate) solution at 400 xG for 30 min.

T and B cells were separated by rosette method with neuramidase-treatedsheep red blood cell.

The E-rosette--negative fraction was treated with appropriately dilutedanti-Leu-i for 30 min. on ice. Treated cells were incubated with newbornrabbits sera used as a complement. B cell blasts activated with PWM at0.25% plus irradiated T cells were used.

B cell blasts (5×10⁴ →1×10⁵ /200 μl RPMI 1640+10% FBS/well) werecultured with various concentrations of BCDF for 5 days.

The concentrations of IgG in culture supernatants were quantitated by asandwich enzyme-linked immunosorbent assay (ELISA).

The result showed that recombinant BSF-2 induced Ig secretion inPWM-stimulated B blast cells (FIG. 25).

EXAMPLE 24

SRBC was administered intravenously in vivo to C3H/Hej mice of 6 weekage in a dose of 1×10⁸ /head and the spleen cells were isolated 5 daysafter. The spleen cells (5×10⁵ /well) and SRBC (1×10⁵ /well) wereincubated for 4 days together with BCDF in the aforesaid concentration.After completion of the incubation, the cells were recovered and thecount of SRBC-specified antibody-producing cells was measured by PFCmethod using a Cunningham chamber.

As shown in FIG. 26, BCDF augmented production of the SRBC-specificantibody-producing cells from the SRBC-sensitized spleen cells singly.

EXAMPLE 25

5FU was administered intravenously to DBA/2 mice in a dose of 0.5mg/head for consecutive 3 days and SRBC was administered intravenouslyin vivo on Day 3 in a dose of 1×10⁸ /head. BCDF was subcutaneously givenfor 3 consecutive days from 2 days after the administration of SRBC in adose of 0.01 to 1.0 μg/head. Sera were subjected to sampling 5 daysafter the administration of SRBC. The concentration of SRBC-specificantibody in serum was measured by a SRBC agglutination test using a 96well microplate. As an agglutination titer, the reciprocal number ofdilution magnification was used.

As shown in FIG. 27, the administratin of BCDF accelerated in a dosedependent manner antibody productivity reduced by the administration of5-FU.

EXAMPLE 26

Cyclophosphamide (CY) was intraperitoneally administered to DBA/2 miceof 6 weeks age in a dose of 4 mg/head (day 0). BCDF was thenadministered subcutaneously and intraperitoneally every day (day 0-day6). On Day 7 after the CY administration, the spleen cells were isolatedand 7.5×10⁴ of the cells were incubated in a soft agar medium for 6 daystogether with 10% PWM-stimulated spleen cell supernatant. Thus thenumber of clusters was measured. For control, mouse serum albumin (MSA)was administered. The results are shown in FIG. 28.

The number of the clusters from spleen cells formed by the addition ofPWM-stimulated spleen cell supernatant increased depending upon the doseof BCDF and reached 4 times at the maximum in the group administeredwith BCDF, as compared to the group administered with MSA.

It is assumed that the in vivo administration of BCDF would stimulatethe growth of hematopoietic stem cells in the spleen.

Many modifications and variations of the present invention are possiblein light of the above teachings. Therefore, it is to be understood thatwithin the scope of the appended claims, the invention may be practicedotherwise than as specifically described.

What is claimed as new and desired to be secured by Letters Patent ofthe United States is:
 1. A method for stimulating bone marrowproliferation comprising:administering to a subject in need thereof aneffective amount of a non-glycosylated polypeptide having human B-celldifferentiation factor activity, said polypeptide having PRO or ALA PROas the N-terminal amino acid(s) and MET as the C-terminal amino acid anda formula selected from the group consisting of amino acid formula (I),(II), (III), and (IV).
 2. A method of differentiating myelomonociticleukemia cells comprising:administering to a subject in need thereof aneffective amount of a non-glycosylated polypeptide having human B-celldifferentiation factor activity, said polypeptide having PRO or ALA PROas the N-terminal amino acid(s) and MET as the C-terminal amino acid anda formula selected from the group consisting of amino acid formula (I),(II), (III), and (IV).